CD20-binding immunotoxins for inducing cellular internalization and methods using same

ABSTRACT

The present invention provides CD20-binding proteins that bind to and rapidly internalize CD20 antigens from a cell surface location to the interior of a cell. CD20-binding proteins of the invention comprise a CD20 binding region and a Shiga toxin effector region. Certain of the disclosed CD20-binding proteins kill cells that express CD20 on their surface. Further, the presently disclosed CD20-binding proteins can comprise additional exogenous materials and are capable of targeted delivery of these additional exogenous materials into the interior of CD20 expressing cells. Such additional materials may include peptides, antigens, enzymes, and polynucleotides. These CD20-binding proteins have uses in methods of internalizing themselves, targeted killing of CD20 expressing cells, delivering exogenous materials into CD20 expressing cells, and treating a variety of diseases involving CD20 expressing cells, such as cancers and immune disorders.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 24, 2014, isnamed 13-03PCT SL.txt and is 170,964 bytes in size.

FIELD OF THE INVENTION

The present invention relates to CD20-binding proteins with the abilityof binding to and forcing the rapid internalization of CD20 antigensfrom a cell surface location to the cell interior. These CD20-bindingproteins have uses as therapeutic molecules for treatment of a varietyof diseases, including cancer and immune disorders.

BACKGROUND OF THE INVENTION

An immunotoxin is a chimeric molecule which combines a cell surfacebinding region, such as from an immunoglobulin domain, and a toxinregion typically derived from a naturally occurring protein toxin, suchas those found in bacteria or plants. The potency of an immunotoxingreatly depends on its efficiency in transiting from the cell surface tothe cytosol, a process that begins with cell internalization (see PirieC et al., J Biol Chem 286: 4165-72 (2011)).

CD20 is a member of a family of polypeptides known as themembrane-spanning 4A (MS4A) family that includes at least 26 proteins inhumans and mice (Ishibashi K et al., Gene 264: 87-93 (2001)). As withall MS4A members, the CD20 sequence predicts three hydrophobic regionsforming a transmembrane molecule that spans the membrane four times, astructural characteristic believed central to its function. Alsopredicted is a single extracellular loop between the proposed third andfourth transmembrane domains and intracellular amino- andcarboxy-terminal regions (Tedder T et al., Proc Natl Acad Sci 85: 208-12(1988)). It is within this extracellular loop of approximately 40 aminoacids that the majority of anti-CD20 monoclonal antibodies (mAbs), suchas rituximab, are believed to bind with alanine-170 and proline-172being the most critical residues. A crystal structure of an antibodybinding a peptide fragment of CD20 using amino acids 163-187 of CD20 hasconfirmed amino acids 170 (alanine) through amino acids 173 (serine) asantigen-antibody interaction points for rituximab and CD20 (Du J et al.,J Biol Chem 282: 15073-80 (2007)).

CD20 is believed to be present on the cell surface as a homo-multimer,likely a tetramer, and electron microscopy has shown that 90% ofcomplexed CD20 is present in the membrane in lipid rafts and microvilli(Li H et al., J Biol Chem 279: 19893-901 (2004)). Lipid rafts aremicro-domains found in the plasma membrane which have high polypeptide,sphingolipid, and cholesterol concentrations (Brown D, London E, AnnuRev Cell Dev Biol 14: 111-36 (1998)). Microvilli, or microvillarchannels, are cell extensions from the plasma membrane surface (Reaven Eet al., J Lipid Res 30: 1551-60 (1989)). Some antibodies to CD20 areknown to bind only when the molecule is present in lipid rafts, such asFMC7 (Polyak M et al., Leukemia 17:1384-89 (2003)) and others, such asrituximab, are known to increase association of CD20 to rafts (Li H etal, supra). It is hypothesized that raft association is important to theproposed function of CD20 as an amplifier of calcium signals that aretransduced through the B-cell antigen receptor (BCR), another proteincommonly located within lipid rafts and found associated with CD20multimers (Polyak M et al., J Biol Chem 283: 18545-52 (2008)).

Antibody-based therapies targeting a CD20 antigen are numerous (seeBoross P, Leusen J, Am J Cancer Res 2: 676-90 (2012), for review). Oneof the attractive characteristics of CD20 as a target for therapiesbased on a mechanism in which the therapeutic remains on the cellsurface in order to function is the lack of CD20 cellularinternalization after being bound by antibody-based therapeutics(Anderson K et al., Blood 63: 1424-33 (1984); Press 0 et al., Blood 69:584-91 (1987)). Although this has proven to be both cell-type andantibody-type specific, in general, CD20 appears to internalize at amuch lower rate than do other cell surface antigens (Beers S et al., SemHematol 47: 107-14 (2010)).

There is a question in the art as to the utility of CD20 antigens as atarget for therapies that require the therapeutic to internalize into atarget cell after binding in order to be effective because of thegeneral finding that CD20 does not readily internalize (Anderson K etal., Blood 63: 1424-33 (1984); Press 0 et al., Blood 69: 584-91 (1987);Beers S et al., Sem Hematol 47: 107-14 (2010)). Thus, there is anunsolved problem in targeting CD20 antigens with immunoglobulin-typetherapeutics that require cell internalization for efficacy—how to forcethe CD20 bound therapeutic into the target cell's interior afterbinding. For example, therapies based on the delivery of an immunotoxinthat targets a CD20 antigen are predicted to be ineffective based oninsufficient CD20 internalization efficiency. Thus, there is a need inthe art to develop effective compositions, therapeutics, and therapeuticmethods that target cell-surface antigens which do not nativelyinternalize at an efficient rate or upon binding by animmunoglobulin-type domain, like CD20.

In particular, there remains a need in the art to identify and developCD20-targeted compositions that trigger rapid and efficient cellularinternalization of the complex of the composition bound to CD20. Forexample, cytotoxic CD20-binding proteins comprising toxin-derivedregions that induce cellular internalization of native CD20 moleculesare desirable for the development of effective cancer andimmuno-modulatory therapeutic molecules that target cells of B-celllineages.

SUMMARY OF THE INVENTION

The present invention provides various CD20-binding proteins forinducing rapid cellular internalization of CD20, which comprise 1) aCD20 binding region, such as an immunoglobulin domain, and 2) a Shigatoxin effector region, such as a truncation of SLT-1A. Upon binding aCD20 antigen on the surface of a cell, the CD20-binding proteins of theinvention are capable of inducing rapid cellular internalization of thecomplex comprising of the CD20-binding protein and a CD20 antigen intothe interior of a eukaryotic cell. The linking of CD20 binding regionswith Shiga-toxin-Subunit-A-derived polypeptides enables the engineeringof cytotoxic Shiga-toxin based molecules that are capable of inducingrapid cellular internalization of natively expressed CD20, as well ascapable of delivering additional exogenous materials into the interiorof CD20 expressing cells. The CD20-binding proteins of the inventionhave uses, e.g., for targeted killing of CD20 positive cell types,delivering exogenous materials, as diagnostic agents, and astherapeutics for the treatment of a variety of conditions in patientssuch as cancers, tumors, and immune disorders related to B-celllineages.

A CD20-binding protein of the invention comprises (a) a CD20 bindingregion comprising an immunoglobulin-type binding region and capable ofspecifically binding an extracellular part of CD20 and (b) a Shiga toxineffector region comprising a polypeptide derived from the amino acidsequence of the A Subunit of at least one member of the Shiga toxinfamily; whereby upon administration of the CD20-binding protein to acell expressing CD20 on a cellular surface, the CD20-binding protein iscapable of inducing rapid cellular internalization of a protein complexcomprising the CD20-binding protein bound to CD20.

For certain embodiments of the CD20-binding proteins of the presentinvention, the CD20 binding region comprises an immunoglobulin-typebinding region comprising a polypeptide selected from the groupconsisting of: a complementary determining region 3 fragment,constrained FR3-CDR3-FR4 polypeptide, single-domain antibody fragment,single-chain variable fragment, antibody variable fragment,antigen-binding fragment, Fd fragment, fibronectin-derived 10^(th)fibronectin type III domain, tenascin type III domain, ankyrin repeatmotif domain, low-density-lipoprotein-receptor-derived A-domain,lipocalin, Kunitz domain, Protein-A-derived Z domain, gamma-Bcrystalline-derived domain, ubiquitin-derived domain, Sac7d-derivedpolypeptide, Fyn-derived SH2 domain, engineered antibody mimic, and anygenetically manipulated counterparts of any of the foregoing that retainCD20 binding functionality.

For certain embodiments, the CD20-binding proteins are capable ofinducing rapid cellular internalization of a CD20 natively present onthe surface of a cell. In certain further embodiments, the CD20-bindingproteins are capable of inducing, in less than about one hour, cellularinternalization of a CD20 natively present on the surface of a cell. Incertain further embodiments, the CD20-binding proteins are capable ofinducing, in less than about one hour, cellular internalization of aCD20 natively present on the surface of a member of a B-cell lineage.

For certain embodiments, upon administration of the CD20-binding proteinto a cell which expresses CD20 on a cellular surface, the CD20-bindingprotein is capable of causing the death of the cell. In certain otherembodiments, the CD20-binding proteins comprise Shiga toxin effectorregions that lack catalytic activity and are not capable of causing thedeath of a cell.

For certain embodiments, upon administration of the CD20-binding proteinto a first population of cells whose members express CD20, and a secondpopulation of cells whose members do not express CD20, the cytotoxiceffect of the CD20-binding protein to members of the first population ofcells relative to members of the second population of cells is at least3-fold greater.

For certain embodiments, the CD20-binding proteins comprise the Shigatoxin effector region comprising or consisting essentially of aminoacids 75 to 251 of SEQ ID NO:1, SEQ ID NO:25, or SEQ ID NO:26. Furtherembodiments are CD20-binding proteins in which the Shiga toxin effectorregion comprises or consists essentially of amino acids 1 to 241 of SEQID NO:1, SEQ ID NO:25, or SEQ ID NO:26; amino acids 1 to 251 of SEQ IDNO:1, SEQ ID NO:25, or SEQ ID NO:26; and/or amino acids 1 to 261 of SEQID NO:1, SEQ ID NO:25, or SEQ ID NO:26.

For certain embodiments, the CD20-binding protein comprises or consistsessentially of amino acids of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:14,or SEQ ID NO:16.

In certain embodiments, the CD20-binding proteins comprise the CD20binding region comprising: (a) a heavy chain variable domain comprisingHCDR1, HCDR2, and HCDR3 amino acid sequences as shown in SEQ ID NO:6,SEQ ID NO:7, and SEQ ID NO:8, respectively, and a light chain variabledomain comprising LCDR1, LCDR2, and LCDR3 amino acid sequences as shownin SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; (b) aheavy chain variable domain comprising HCDR1, HCDR2, and HCDR3 aminoacid sequences as shown in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23,respectively, and a light chain variable domain comprising LCDR1, LCDR2,and LCDR3 amino acid sequences as shown in SEQ ID NO:24, SEQ ID NO:10,and SEQ ID NO:11, respectively; or (c) a heavy chain variable (VH)domain comprising HCDR1, HCDR2, and HCDR3 amino acid sequences as shownin SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:27, respectively, and alight chain variable (VL) domain comprising LCDR1, LCDR2, and LCDR3amino acid sequences as shown in SEQ ID NO:28, SEQ ID NO:10, and SEQ IDNO:29, respectively. Further embodiments are CD20-binding proteinscomprising the immunoglobulin-type binding region comprising orconsisting essentially of amino acids 2-245 of SEQ ID NO:4. Furtherembodiments are CD20-binding proteins comprising the immunoglobulin-typebinding region comprising or consisting essentially of amino acids 2-245of SEQ ID NO:4 and the Shiga toxin effector region comprising orconsisting essentially of amino acids 75-251 of SEQ ID NO:1. Furtherembodiments are CD20-binding proteins comprising or consistingessentially of SEQ ID NO:4 or SEQ ID NO:16.

In certain embodiments, the CD20-binding proteins comprise Shiga toxineffector regions which comprise a mutation relative to a naturallyoccurring A Subunit of a member of the Shiga toxin family which changesthe enzymatic activity of the Shiga toxin effector region, the mutationselected from at least one amino acid residue deletion or substitution.

Certain embodiments of the CD20-binding proteins can also be utilizedfor the delivery of additional exogenous material into a cell thatexpresses CD20 on a cellular surface. These embodiments comprise a CD20binding region comprising (a) an immunoglobulin-type polypeptide capableof specifically binding an extracellular part of a CD20 molecule, (b) aShiga toxin effector region comprising a polypeptide derived from theamino acid sequence of at least one member of the Shiga toxin family,and (c) an additional exogenous material; whereby upon administration ofthe CD20-binding protein to a cell expressing CD20 on a cellularsurface, the CD20-binding protein is capable of inducing rapid cellularinternalization of a protein complex comprising the CD20-binding proteinbound to CD20 and capable of delivering the additional exogenousmaterial into the interior of the cell. In certain further embodiments,the CD20-binding proteins comprise the CD20 binding region comprising:(a) a heavy chain variable domain comprising HCDR1, HCDR2, and HCDR3amino acid sequences as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ IDNO:8, respectively, and a light chain variable domain comprising LCDR1,LCDR2, and LCDR3 amino acid sequences as shown in SEQ ID NO:9, SEQ IDNO:10, and SEQ ID NO:11, respectively; or (b) a heavy chain variabledomain comprising HCDR1, HCDR2, and HCDR3 amino acid sequences as shownin SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, respectively, and alight chain variable domain comprising LCDR1, LCDR2, and LCDR3 aminoacid sequences as shown in SEQ ID NO:24, SEQ ID NO:10, and SEQ ID NO:11,respectively; or (c) a heavy chain variable (VH) domain comprisingHCDR1, HCDR2, and HCDR3 amino acid sequences as shown in SEQ ID NO:21,SEQ ID NO:22, and SEQ ID NO:27, respectively, and a light chain variable(VL) domain comprising LCDR1, LCDR2, and LCDR3 amino acid sequences asshown in SEQ ID NO:28, SEQ ID NO:10, and SEQ ID NO:29, respectively.

In certain embodiments, the additional exogenous material is selectedfrom the group consisting of peptides, polypeptides, proteins, andpolynucleotides. In certain embodiments, the additional exogenousmaterial comprises a protein or polypeptide comprising an enzyme. Incertain other embodiments, the additional exogenous material is anucleic acid, such as, e.g. a ribonucleic acid that functions as a smallinhibiting RNA (siRNA) or microRNA (miRNA).

In certain embodiments, the additional exogenous material is a peptideand the peptide is an antigen. In certain embodiments, the additionalexogenous material is an antigen derived from a bacterial protein. Incertain other embodiments, the antigen is derived from a protein mutatedin cancer. Further embodiments are ones in which the antigen is derivedfrom a protein aberrantly expressed in cancer. Still further embodimentsare ones in which the antigen is derived from a T-cell complementarydetermining region.

For certain embodiments, the antigen is included within the CD20-bindingprotein as part of a polypeptide fusion in which the peptide antigen islocated between the binding region and the toxin effector region of asingle-chain protein. In certain embodiments, the additional exogenousmaterial is an antigen derived from a viral protein. In certainembodiments, the antigen comprises or consists essentially of SEQ IDNO:3, the influenza Matrix 58-66 antigen. In certain furtherembodiments, the CD20-binding protein comprises or consists essentiallyof SEQ ID NO:16.

The invention also includes pharmaceutical compositions comprising aCD20-binding protein of the present invention and at least onepharmaceutically acceptable excipient or carrier; and the use of such acytotoxic protein or a composition comprising it in methods of theinvention as further described herein.

The present invention also provides polynucleotides that encode theCD20-binding proteins of the invention, expression vectors that comprisethe polynucleotides of the invention, as well as host cells comprisingthe expression vectors of the invention.

Additionally, the present invention provides a method of rapidlyinducing cellular internalization of a CD20-binding protein of thepresent invention into a CD20 expressing cell(s), the method comprisingthe step of contacting the cell(s) with a CD20-binding protein of thepresent invention or a pharmaceutical composition thereof. Similarly,the present invention provides a method of internalizing a cell surfacelocalized CD20 bound by a CD20-binding protein in a patient, the methodcomprising the step of administering to the patient a CD20-bindingprotein or pharmaceutical composition of the present invention.

Additionally, the present invention provides a method of killing a CD20expressing cell(s) comprising contacting the cell(s), either in vitro orin vivo, with a CD20-binding protein or pharmaceutical composition ofthe present invention.

Additionally, the present invention provides a method for deliveringexogenous material to the inside of a cell(s) comprising contacting thecell(s), either in vitro or in vivo, with a CD20-binding protein orpharmaceutical composition of the present invention.

The present invention further provides a method for delivering exogenousmaterial to the inside of a cell(s) in a patient, wherein the cellexpresses CD20 on its surface, the method comprising the step ofadministering to the patient a CD20-binding protein of the presentinvention.

Additionally, the present invention provides methods of killing cellscomprising the step of contacting the cell with a CD20-binding proteinof the invention or a pharmaceutical composition of the invention. Incertain embodiments of the cell killing method, the step of contactingthe cell(s) occurs in vitro. In certain other embodiments, the step ofcontacting the cell(s) occurs in vivo.

Also, the present invention provides a method of treating a disease,disorder, or condition in patients comprising the step of administeringto a patient in need thereof a therapeutically effective amount of aCD20-binding protein of the invention or a pharmaceutical composition ofthe invention. In certain embodiments of the treating method, thedisease, disorder, or condition to be treated using this method of theinvention involves a cell(s) or cell type(s) which express CD20 on acellular surface, such as, e.g., a cancer cell, a tumor cell, or animmune cell. A further embodiment is a method of treating a diseaseinvolving a cancer or tumor cell associated with the disease selectedfrom the group consisting of: bone cancer, leukemia, lymphoma, melanoma,or myeloma. In certain embodiments of this method, the disorder is animmune disorder associated with a disease selected from the groupconsisting of: amyloidosis, ankylosing spondylitis, asthma, Crohn'sdisease, diabetes, graft rejection, graft-versus host disease,Hashimoto's thyroiditis, hemolytic uremic syndrome, HIV-relateddiseases, lupus erythematosus, multiple sclerosis, polyarteritis,psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma,septic shock, Sjörgren's syndrome, ulcerative colitis, and vasculitis.

Among certain embodiments of the present invention is the use of aCD20-binding protein of the invention in the manufacture of a medicamentfor the treatment or prevention of a cancer or immune disorder. Amongcertain embodiments of the present invention is a cytotoxic protein or apharmaceutical composition comprising said protein for use in thetreatment or prevention of a cancer, tumor, or immune disorder.

These and other features, aspects and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying figures. Theaforementioned elements of the invention may be combined or removedfreely in order to make other embodiments, without any statement toobject such combination or removal hereinafter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the general architecture of exemplary CD20-binding proteinsof the present invention.

FIG. 2 graphically shows the change in total body luminescence with theadministration of αCD20scFv::SLT-1A version 1 and αCD20scFv::SLT-1Aversion 2 in a disseminated Raji-luc xenograft model.

FIG. 3 graphically shows the increased survival of Raji-luc xenograftmodel mice with the administration of αCD20scFv::SLT-1A version 1 andαCD20scFv::SLT-1A version 2.

FIG. 4 graphically shows the change in tumor volume with theadministration of αCD20scFv::SLT-1A version 1 and αCD20scFv::SLT-1Aversion 2 in a Raji subcutaneous xenograft model.

FIG. 5 shows B-cell depletion in a non-human primate study with theadministration of αCD20scFv::SLT-1A version 1. Specifically, the subsetsof CD20+ B-cells that expressed CD21 were analyzed.

FIG. 6 shows B-cell depletion in a non-human primate study with theadministration of αCD20scFv::SLT-1A version 1. Specifically, the subsetsof CD20+ B-cells that did not express CD21 were analyzed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is described more fully hereinafter usingillustrative, non-limiting embodiments, and references to theaccompanying figures. This invention may, however, be embodied in manydifferent forms and should not be construed as to be limited to theembodiments set forth below. Rather, these embodiments are provided sothat this disclosure is thorough and conveys the scope of the inventionto those skilled in the art.

In order that the present invention may be more readily understood,certain terms are defined below. Additional definitions may be foundwithin the detailed description of the invention.

As used in the specification and the appended claims, the terms “a,”“an” and “the” include both singular and the plural referents unless thecontext clearly dictates otherwise.

As used in the specification and the appended claims, the term “and/or”when referring to two species, A and B, means at least one of A and B.As used in the specification and the appended claims, the term “and/or”when referring to greater than two species, such as A, B, and C, meansat least one of A, B, or C, or at least one of any combination of A, B,or C (with each species in singular or multiple possibility).

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer (or components) or group of integers (or components),but not the exclusion of any other integer (or components) or group ofintegers (or components).

Throughout this specification, the term “including” is used to mean“including but not limited to.” “Including” and “including but notlimited to” are used interchangeably.

The term “amino acid residue” or “amino acid” includes reference to anamino acid that is incorporated into a protein, polypeptide, or peptide.The term “polypeptide” includes any polymer of amino acids or amino acidresidues. The term “polypeptide sequence” refers to a series of aminoacids or amino acid residues from which a polypeptide is physicallycomposed. A “protein” is a macromolecule comprising one or morepolypeptides chains. A “peptide” is a small polypeptide of sizes lessthan a total of 15-20 amino acid residues.

The terms “amino acid,” “amino acid residue,” or polypeptide sequenceinclude naturally occurring amino acids and, unless otherwise limited,also include known analogs of natural amino acids that can function in asimilar manner as naturally occurring amino acids. The amino acidsreferred to herein are described by shorthand designations as follows inTable A:

TABLE A Amino Acid Nomenclature Name 3-letter 1-letter Alanine Ala AArginine Arg R Asparagine Asn N Aspartic Acid or Aspartate Asp DCysteine Cys C Glutamic Acid or Glutamate Glu E Glutamine Gln Q GlycineGly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys KMethionine Met M Phenylalanine Phe F Proline Pro P Serine Ser SThreonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

The phrase “conservative substitution” with regard to a polypeptide,refers to a change in the amino acid composition of the polypeptide thatdoes not substantially alter the function and structure of the overallpolypeptide (see Creighton, Proteins: Structures and MolecularProperties (W. H. Freeman and Company, New York (2nd ed., 1992)).

As used herein, the term “expressed,” “expressing” or “expresses” refersto translation of a polynucleotide or nucleic acid into a polypeptide orprotein. The expressed polypeptide or protein may remain intracellular,become a component of the cell surface membrane or be secreted into anextracellular space.

As used herein, the symbol “a” is shorthand for an immunoglobulin-typebinding region capable of binding to the biomolecule following thesymbol. The symbol “a” is used to refer to the functional characteristicof an immunoglobulin-type binding region based on its capability ofbinding to the biomolecule following the symbol.

The term “selective cytotoxicity” with regard to the cytotoxic activityof a CD20-binding protein refers to the relative levels of cytotoxicitybetween a targeted cell population and a non-targeted bystander cellpopulation, which can be expressed as a ratio of the half-maximalcytotoxic concentration (CD₅₀) for a targeted cell type over the CD₅₀for an untargeted cell type to show preferentiality of cell killing ofthe targeted cell type.

For purposes of the present invention, the term “effector” meansproviding a biological activity, such as cytotoxicity, biologicalsignaling, enzymatic catalysis, subcellular routing, and/orintermolecular binding resulting in the recruit of a factors and/orallosteric effects.

For purposes of the present invention, the phrase “derived from” meansthe polypeptide region comprises amino acid sequences originally foundin a protein and may now comprise additions, deletions, truncations, orother alterations form the original sequence such that overall functionand structure are substantially conserved.

Introduction

The present invention solves problems for engineering therapeuticstargeting CD20 that require cell internalization for function becauseShiga-toxin-Subunit-A derived effector regions induce the cellularinternalization of CD20. The present invention provides CD20-bindingproteins that bind to extracellular CD20 antigens and rapidlyinternalize CD20 from a cell membrane location to the interior of acell. Certain of the disclosed CD20-binding proteins kill cells whichexpress CD20 on their surface. Certain of the disclosed CD20-bindingproteins are capable of precisely delivering additional exogenousmaterial in the form of molecular cargos to the interior of cells whichexpress CD20 on their surface. The present invention expands theuniverse of immunotoxin-drugable targets to include CD20 and enables theprecise delivery of payloads to the interiors of CD20 expressing cells.

I. The General Structure of the CD20-Binding Proteins of the Invention

The present invention provides various CD20-binding proteins for theselective killing of specific cell types, each CD20-binding proteincomprising 1) a CD20 binding region comprising immunoglobulin-typebinding regions for cell targeting and 2) a Shiga toxin effector regionfor cell killing. The linking of CD20 targeting immunoglobulin-typebinding regions with Shiga-toxin-Subunit-A-derived regions enables theengineering of cell-type specific targeting of the potent Shiga toxincytotoxicity. This system is modular, in that various Shiga toxineffector regions and additional exogenous materials may be linked to thesame CD20 binding region to provide diverse applications involving CD20expressing cells. CD20-binding proteins of the invention comprise Shigatoxin effector regions derived from the A Subunits of members of theShiga toxin family linked to immunoglobulin-type CD20 binding regionswhich can bind specifically to at least one extracellular part of a CD20molecule spanning the outer cell membrane of a eukaryotic cell. Thisgeneral structure is modular in that various CD20 binding regions can belinked to Shiga-toxin-Subunit-A derived effector regions at variouspositions or with different linkers between them to produce variationsof the same general structure (see e.g. FIG. 1).

A. CD20 Binding Regions Comprising an Immunoglobulin-Type Binding Region

For purposes of the present invention, the term “CD20 binding region”refers to a polypeptide region capable of specifically binding anextracellular part of a CD20 molecule. While the name CD20 might referto multiple proteins with related structures and polypeptide sequencesfrom various species, for the purposes of the present invention the term“CD20” refers to the B-lymphocyte antigen CD20 proteins present inmammals whose exact sequence might vary slightly based on the isoformand from individual to individual. For example, in humans CD20 refers tothe protein represented by the predominant polypeptide sequence UnitProtP11836 and NCBI accession NP 690605.1; however, different isoforms andvariants may exist. The polypeptide sequence of various CD20 proteinshas been described in various species, such as bats, cats, cattle, dogs,mice, marmosets, and rats, and can be predicted by bioinformatics innumerous other species based on genetic homology (e.g. CD20 has beenpredicted in various primates, including baboons, macaques, gibbons,chimpanzees, and gorillas) (see Zuccolo J et al., PLoS One 5: e9369(2010) and NCBI protein database (National Center for BiotechnologyInformation, U.S.). A skilled worker will be able to identify a CD20protein in mammals, even if it differs from the referenced sequencesslightly.

An extracellular part of a CD20 molecule refers to a portion of itsstructure exposed to the extracellular environment when the CD20molecule is natively present in a cell membrane. In this context,exposed to the extracellular environment means that part of the CD20molecule is accessible by, e.g., an antibody or at least a bindingmoiety smaller than an antibody such as a single-domain antibody domain,a nanobody, a heavy-chain antibody domain derived from camelids orcartilaginous fishes, a single-chain variable fragment, or any number ofengineered alternative scaffolds to immunoglobulins (see below). Theexposure of a part of CD20 may be empirically determined by the skilledworker using methods known in the art. Note that some portion of CD20,which was predicted not to be accessible to an antibody in theextracellular space based on its location within CD20, was empiricallyshown to be accessible by a monoclonal antibody (Teeling J et al., J.Immunol. 177: 362-71 (2006)).

CD20 binding regions are commonly derived from antibody or antibody-likestructures; however, alternative scaffolds from other sources arecontemplated within the scope of the term. In certain embodiments, theCD20 binding region is derived from an immunoglobulin-derived bindingregion, such as an antibody paratope. In certain other embodiments, theCD20 binding region comprises an immunoglobulin-type binding region thatis an engineered polypeptide not derived from any immunoglobulin domain.There are numerous immunoglobulin-derived binding regions contemplatedas components in the present invention.

CD20-binding proteins of the invention comprise an immunoglobulin-typebinding region comprising one or more polypeptides capable ofselectively and specifically binding an extracellular part of CD20. Theterm “immunoglobulin-type binding region” as used herein refers to apolypeptide region capable of binding one or more target biomolecules,such as an antigen or epitope. Immunoglobulin-type binding regions arefunctionally defined by their ability to bind to target molecules, andall the immunoglobulin-type binding regions of the present invention arecapable of binding CD20. Immunoglobulin-type binding regions arecommonly derived from antibody or antibody-like structures; however,alternative scaffolds from other sources are contemplated within thescope of the term.

Immunoglobulin (Ig) proteins have a structural domain known as an Igdomain. Ig domains range in length from about 70-110 amino acid residuesand possess a characteristic Ig-fold, in which typically 7 to 9antiparallel beta strands arrange into two beta sheets which form asandwich-like structure. The Ig fold is stabilized by hydrophobic aminoacid interactions on inner surfaces of the sandwich and highly conserveddisulfide bonds between cysteine residues in the strands. Ig domains maybe variable (IgV or V-set), constant (IgC or C-set) or intermediate (IgIor I-set). Some Ig domains may be associated with a complementaritydetermining region (CDR), also referred to as antigen binding region(ABR), which is important for the specificity of antibodies binding totheir epitopes. Ig-like domains are also found in non-immunoglobulinproteins and are classified on that basis as members of the Igsuperfamily of proteins. The HUGO Gene Nomenclature Committee (HGNC)provides a list of members of the Ig-like domain containing family.

An immunoglobulin-type binding region may be a polypeptide sequence ofantibody or antigen-binding fragment thereof wherein the amino acidsequence has been varied from that of a native antibody or an Ig-likedomain of a non-immunoglobulin protein, for example by molecularengineering or selection by library screening. Because of the relevanceof recombinant DNA techniques and in vitro library screening in thegeneration of immunoglobulin-type binding regions, antibodies can beredesigned to obtain desired characteristics, such as smaller size, cellentry, or other therapeutic improvements. The possible variations aremany and may range from the changing of just one amino acid to thecomplete redesign of, for example, a variable region. Typically, changesin the variable region will be made in order to improve theantigen-binding characteristics, improve variable region stability, orreduce the potential for immunogenic responses.

There are numerous immunoglobulin-type binding regions that bind anextracellular part of CD20 contemplated in the present invention. Incertain embodiments, the immunoglobulin-type binding region is derivedfrom an immunoglobulin binding region, such as an antibody paratopecapable of binding an extracellular part of CD20. In certain otherembodiments, the immunoglobulin-type binding region comprises anengineered polypeptide not derived from any immunoglobulin domain butthat functions like an immunoglobulin binding region by providinghigh-affinity binding to an extracellular part of CD20. This engineeredpolypeptide may optionally include polypeptide scaffolds comprising orconsisting essentially of complementary determining regions fromimmunoglobulins as described herein.

There are numerous immunoglobulin-derived binding regions andnon-immunoglobulin engineered polypeptides in the prior art that areuseful for targeting the CD20-binding proteins of the invention to CD20expressing cells. In certain embodiments, the immunoglobulin-typebinding region of the present CD20-binding proteins is selected from thegroup which includes single-domain antibody domains (sdAb), nanobodies,heavy-chain antibody domains derived from camelids (VHH fragments),bivalent nanobodies, heavy-chain antibody domains derived fromcartilaginous fishes, immunoglobulin new antigen receptors (IgNARs),VNAR fragments, single-chain variable (scFv) fragments, multimerizingscFv fragments (diabodies, triabodies, tetrabodies), bispecific tandemscFv fragments, disulfide stabilized antibody variable (Fv) fragments,disulfide stabilized antigen-binding (Fab) fragments consisting of theV_(L), V_(H), C_(L) and C_(H) 1 domains, divalent F(ab′)2 fragments, Fdfragments consisting of the heavy chain and C_(H)1 domains, single chainFv-C_(H)3 minibodies, bispecific minibodies, dimeric C_(H)2 domainfragments (C_(H)2D), Fc antigen binding domains (Fcabs), isolatedcomplementary determining region 3 (CDR3) fragments, constrainedframework region 3, CDR3, framework region 4 (FR3-CDR3-FR4)polypeptides, small modular immunopharmaceutical (SMIP) domains, and anygenetically manipulated counterparts of the foregoing that retain itsparatope and binding function (see, Weiner L, Cell 148: 1081-4 (2012);Ahmad Z et al., Clin Dev Immunol 2012: 980250 (2012), for reviews).

In accordance with certain other embodiments, the immunoglobulin-typebinding region of the CD20-binding proteins of the invention may includeengineered, alternative scaffolds to immunoglobulin domains that exhibitsimilar functional characteristics, such as high-affinity and specificbinding to CD20, and enable the engineering of improved characteristics,such as greater stability or reduced immunogenicity. For certainembodiments of the CD20-binding proteins of the invention, theimmunoglobulin-type binding region is selected from the group whichincludes engineered, fibronectin-derived, 10^(th) fibronectin type III(10Fn3) domains (monobodies, AdNectins™, or AdNexins™); engineered,tenascin-derived tenascin type III domains (Centryns™); engineered,ankyrin repeat motif containing polypeptides (DARPins™); engineered,low-density-lipoprotein-receptor-derived, A domains (LDLR-A) (Avimers™);lipocalins (anticalins); engineered, protease inhibitor-derived, Kunitzdomains; engineered, Protein-A-derived, Z domains (Affibodies™);engineered, gamma-B crystalline-derived scaffolds or engineered,ubiquitin-derived scaffolds (Affilins); Sac7d-derived polypeptides(Nanoffitins® or affitins); engineered, Fyn-derived, SH2 domains(Fynomers®); and engineered antibody mimics; and any geneticallymanipulated counterparts of the foregoing that retains its bindingfunctionality (Wörn A, Plückthun A, J Mol Biol 305: 989-10 (2001); Xu Let al., Chem Biol 9: 933-42 (2002); Wikman M et al., Protein Eng Des Sel17: 455-62 (2004); Binz H et al., Nat Biotechnol 23: 1257-68 (2005);Holliger P, Hudson P, Nat Biotechnol 23: 1126-36 (2005); Gill D, DamleN, Curr Opin Biotech 17: 653-8 (2006); Koide A, Koide S, Methods MolBiol 352: 95-109 (2007)).

Nonlimiting examples of protein constructs encompassed within the term“binding region” as used herein include: (i) an Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH 1 domains; (ii)an F(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) an Fd fragmentconsisting of the VH and CH 1 domains; (iv) an Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAbfragment, which consists of a VH domain; and (vi) an isolated CDR.Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they may be recombinantly joined by asynthetic linker, creating a single protein chain in which the VL and VHdomains pair to form monovalent molecules (known as single chain Fv(scFv)). The most commonly used linker is a 15-residue (Gly4Ser)3peptide, but other linkers are also known in the art. Single chainantibodies are also intended to be encompassed within the term “bindingregion” as used herein.

It is also anticipated that alternative scaffolds that provide bindingfunction are within the scope of the term “binding region” as usedherein. Some examples of the alternative scaffolds include diabodies, aCDR3 peptide, a constrained FR3-CDR3-FR4 peptide, a nanobody (U.S.patent application publication 2008/0107601), a bivalent nanobody, smallmodular immunopharmaceuticals (SMIPs), a shark variable IgNAR domain (WO03/014161), a minibody and any fragment or chemically or geneticallymanipulated counterparts that retain target molecule binding function.

An “antibody-derived sequence” as used herein, means an amino acidsequence of an antibody or antigen-binding fragment thereof wherein theamino acid sequence has been varied from that of a native antibody.Because of the relevance of recombinant DNA techniques in the generationof antibodies, antibodies can be redesigned to obtain desiredcharacteristics. The possible variations are many and range from thechanging of just one or a few amino acids to the complete redesign of,for example, a variable region. Typically, changes in the variableregion will be made in order to improve the antigen-bindingcharacteristics, improve variable region stability, or reduce the riskof immunogenicity.

As used herein, the term “heavy chain variable (VH) domain” or “lightchain variable (VL) domain” respectively refer to any native antibody VHor VL domain (e.g., a human VH or VL domain) as well as any derivativethereof retaining at least qualitative antigen binding ability of thecorresponding native antibody (e.g., a humanized VH or VL domain derivedfrom a native murine VH or VL domain). A VH or VL domain consists of a“framework” region interrupted by the three CDRs. The framework regionsserve to align the CDRs for specific binding to an epitope of anantigen. From amino-terminus to carboxyl-terminus, both VH and VLdomains comprise the following framework (FR) and CDR regions: FR1,CDR1, FR2, CDR2, FR3, CDR3, FR4. The assignment of amino acids to eachdomain is in accordance with the definitions of Kabat, Sequences ofProteins of Immunological Interest (5th ed., National Institutes ofHealth, Bethesda, Md., 1991), or Chothia and Lesk, J. Mol. Biol. 196:901-17 (1987); Chothia et al., Nature 342:878-83, (1989). CDRs 1, 2, and3 of a VH domain are also referred to herein, respectively, as HCDR1,HCDR2, and HCDR3; CDRs 1, 2, and 3 of a VL domain are also referred toherein, respectively, as LCDR1, LCDR2, and LCDR3.

In some embodiments of the CD20-binding proteins of the presentinvention, the binding region comprises an antibody or anantibody-derived sequence that comprises a specific set ofcomplementarity determining regions, or CDRs. CDRs are defined sequenceregions within the variable domains of antibodies that are necessary forspecific binding of the antibody to its antigenic determinants. In oneembodiment of the invention, the set of CDRs comprise three CDRs derivedfrom the heavy chain of the antibody and three CDRs derived from lightchain of the antibody. In some embodiments, the three heavy chain CDRscomprise: (a) a heavy chain variable domain comprising HCDR1, HCDR2, andHCDR3 amino acid sequences as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQID NO:8, respectively, and a light chain variable domain comprisingLCDR1, LCDR2, and LCDR3 amino acid sequences as shown in SEQ ID NO:9,SEQ ID NO:10, and SEQ ID NO:11, respectively; (b) a heavy chain variabledomain comprising HCDR1, HCDR2, and HCDR3 amino acid sequences as shownin SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, respectively, and alight chain variable domain comprising LCDR1, LCDR2, and LCDR3 aminoacid sequences as shown in SEQ ID NO:24, SEQ ID NO:10, and SEQ ID NO:11,respectively; or (c) a heavy chain variable (VH) domain comprisingHCDR1, HCDR2, and HCDR3 amino acid sequences as shown in SEQ ID NO:21,SEQ ID NO:22, and SEQ ID NO:27, respectively, and a light chain variable(VL) domain comprising LCDR1, LCDR2, and LCDR3 amino acid sequences asshown in SEQ ID NO:28, SEQ ID NO:10, and SEQ ID NO:29, respectively.Additionally, in certain embodiments of the invention, the bindingregion comprises or consists essentially of amino acids 2 to 245 of SEQID NO:4.

This system is modular, in that various, diverse immunoglobulin-typebinding regions can be used with the same Shiga toxin effector region totarget different extracellular epitopes of CD20. It will be appreciatedby the skilled worker that any CD20 binding region of an immunoglobulintype capable of binding an extracellular part of CD20 may be used todesign or select an immunoglobulin-type binding region to be linked tothe Shiga toxin effector region to produce a CD20-binding protein of theinvention.

B. Shiga Toxin Effector Regions Derived from a Subunits of Members ofthe Shiga Toxin Family

For purposes of the present invention, the phrase “Shiga toxin effectorregion” refers to a polypeptide region derived from a Shiga toxin ASubunit of a member of the Shiga toxin family that is capable ofinactivating ribosomes and effectuating cytotoxicity and/or cytostaticeffects. A member of the Shiga toxin family refers to any member of afamily of naturally occurring protein toxins which are structurally andfunctionally related, notably toxins isolated from S. dysenteriae and E.coli (Johannes, Nat Rev Microbiol 8: 105-16 (2010)). For example, theShiga toxin family encompasses true Shiga toxin (Stx) isolated from S.dysenteriae serotype 1, Shiga-like toxin 1 variants (SLT1 or Stx1 orSLT-1 or Slt-I) isolated from serotypes of enterohemorrhagic E. coli,and Shiga-like toxin 2 variants (SLT2 or Stx2 or SLT-2) isolated fromserotypes of enterohemorrhagic E. coli. SLT1 differs by only one residuefrom Stx, and both have been referred to as Verocytotoxins or Verotoxins(VTs) (O'Brien, Curr Top Microbiol Immunol 180: 65-94 (1992)). AlthoughSLT1 and SLT2 variants are only about 53-60% similar to each other atthe amino acid sequence level, they share mechanisms of enzymaticactivity and cytotoxicity common to the members of the Shiga toxinfamily (Johannes, Nat Rev Microbiol 8: 105-16 (2010)). Over 39 differentShiga toxins have been described, such as the defined subtypes Stx1a,Stx1c, Stx1d, and Stx2a-g (Scheutz F et al., J Clin Microbiol 50:2951-63 (2012)). Members of the Shiga toxin family are not naturallyrestricted to any bacterial species because Shiga-toxin-encoding genescan spread among bacterial species via horizontal gene transfer (StrauchE et al., Infect Immun 69: 7588-95 (2001); Zhaxybayeva O, Doolittle W,Curr Biol. 21: R242-6 (2011)). As an example of interspecies transfer, aShiga toxin was discovered in a strain of A. haemolyticus isolated froma patient (Grotiuz G et al., J Clin Microbiol 44: 3838-41 (2006)). Oncea Shiga toxin encoding polynucleotide enters a new subspecies orspecies, the Shiga toxin amino acid sequence is presumed to be capableof developing slight sequence variations due to genetic drift and/orselective pressure while still maintaining a mechanism of cytotoxicitycommon to members of the Shiga toxin family (see Scheutz, J ClinMicrobiol 50: 2951-63 (2012)).

Shiga toxin effector regions of the invention comprise or consistessentially of a polypeptide derived from a Shiga toxin A Subunitdissociated from any form of its native Shiga toxin B Subunit. Inaddition, the CD20-binding proteins of the present invention do notcomprise any polypeptide comprising or consisting essentially of afunctional binding domain of a Shiga toxin B subunit. Rather, the Shigatoxin A Subunit derived regions are functionally associated withheterologous CD20 binding regions to effectuate cell targeting to CD20expressing cells.

In certain embodiments, a Shiga toxin effector region of the inventionmay comprise or consist essentially of a full length Shiga toxin ASubunit (e.g. SLT-1A (SEQ ID NO:1), StxA (SEQ ID NO:25), or SLT-2A (SEQID NO:26)), noting that naturally occurring Shiga toxin A Subunits maycomprise precursor forms containing signal sequences of about 22 aminoacids at their amino-terminals which are removed to produce mature Shigatoxin A Subunits. One specific example of a “toxin effector region” isone that is derived from the A chain of Shiga-like toxin 1 (SLT-1) (SEQID NO:1). The A chain of SLT-1 is composed of 293 amino acids with theenzymatic (toxic) domain spanning residues 1 to 239. In otherembodiments, the Shiga toxin effector region of the invention comprisesor consists essentially of a truncated Shiga toxin A Subunit which isshorter than a full-length Shiga toxin A Subunit.

Shiga-like toxin 1 A Subunit truncations are catalytically active,capable of enzymatically inactivating ribosomes in vitro, and cytotoxicwhen expressed within a cell (LaPointe, J Biol Chem 280: 23310-18(2005)). The smallest Shiga toxin A Subunit fragment exhibiting fullenzymatic activity is a polypeptide composed of residues 1-239 of Slt1A(LaPointe, J Biol Chem 280: 23310-18 (2005)). Although the smallestfragment of the Shiga toxin A Subunit reported to retain substantialcatalytic activity was residues 75-247 of StxA (Al-Jaufy, Infect Immun62: 956-60 (1994)), a StxA truncation expressed de novo within aeukaryotic cell requires only up to residue 240 to reach the cytosol andexert catalytic inactivation of ribosomes (LaPointe, J Biol Chem 280:23310-18 (2005)).

Shiga toxin effector regions may commonly be smaller than the fulllength A subunit. It is preferred that the Shiga toxin effector regionmaintain the polypeptide region from amino acid position 77 to 239(SLT-1A (SEQ ID NO:1), StxA (SEQ ID NO:25), or SLT-2A (SEQ ID NO:26)) orthe equivalent in other A Subunits of members of the Shiga toxin family.For example, in certain embodiments of the invention, a Shiga toxineffector region derived from SLT-1A may comprise or consist essentiallyof amino acids 75 to 251 of SEQ ID NO:1, 1 to 241 of SEQ ID NO:1, 1 to251 of SEQ ID NO:1, or amino acids 1 to 261 of SEQ ID NO:1.

Similarly, among certain other embodiments, the Shiga toxin effectorregions derived from StxA may comprise or consist essentially of aminoacids 75 to 251 of SEQ ID NO:25, 1 to 241 of SEQ ID NO:25, 1 to 251 ofSEQ ID NO:25, or amino acids 1 to 261 of SEQ ID NO:25. Additionally,among certain other embodiments, the Shiga toxin effector regionsderived from SLT-2 may comprise or consist essentially of amino acids 75to 251 of SEQ ID NO:26, 1 to 241 of SEQ ID NO:26, 1 to 251 of SEQ IDNO:26, or amino acids 1 to 261 of SEQ ID NO:26.

The invention further provides variants of the CD20-binding proteins ofthe invention, wherein the Shiga toxin effector region differs from anaturally occurring Shiga toxin A Subunit by up to 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25, 30, 35, 40 or more amino acid residues (but by nomore than that which retains at least 85%, 90%, 95%, 99% or more aminoacid sequence identity). Thus, a polypeptide region derived from an ASubunit of a member of the Shiga toxin family may comprise additions,deletions, truncations, or other alterations from the original sequenceso long as at least 85%, 90%, 95%, 99% or more amino acid sequenceidentity is maintained to a naturally occurring Shiga toxin A Subunit.

Accordingly, in certain embodiments, the Shiga toxin effector regioncomprises or consists essentially of amino acid sequences having atleast 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%or 99.7% overall sequence identity to a naturally occurring Shiga toxinA Subunit, such as SLT-1A (SEQ ID NO:1), Stx (SEQ ID NO:25), and/orSLT-2A (SEQ ID NO:26).

Optionally, either a full length or a truncated version of the Shigatoxin A Subunit may comprise one or more mutations (e.g. substitutions,deletions, insertions or inversions). In certain embodiments that arepotently cytotoxic, the Shiga toxin effector region has sufficientsequence identity to retain cytotoxicity after entry into a cell, eitherby well-known methods of host cell transformation, transfection,infection or induction, or by internalization mediated by the celltargeting, immunoglobulin-type binding region linked with the Shigatoxin effector region. The most critical residues for enzymatic activityand/or cytotoxicity in the Shiga toxin A Subunits have been mapped tothe following residue-positions: aspargine-75, tyrosine-77,glutamate-167, arginine-170, and arginine-176 among others (Di, Toxicon57: 535-39 (2011)). In any one of the embodiments of the presentinvention, the Shiga toxin effector region may preferably but notnecessarily maintain one or more conserved amino acids at positions,such as those found at positions 77, 167, 170, and 203 in StxA, SLT-1A,or the equivalent conserved position in other members of the Shiga toxinfamily which are typically required for cytotoxic activity. The capacityof a CD20-binding protein of the invention to cause cell death, e.g. itscytotoxicity, may be measured using any one or more of a number ofassays well known in the art.

In certain embodiments of the invention, one or more amino acid residuesmay be mutated or deleted in order to reduce or eliminate cytotoxicactivity of the Shiga toxin effector region. The cytotoxicity of the ASubunits of members of the Shiga toxin family may be reduced oreliminated by mutation or truncation. The positions labeled tyrosine-77,glutamate-167, arginine-170, tyrosine-114, and tryptophan-203 have beenshown to be important for the catalytic activity of Stx, Stx1, and Stx2(Hovde C et al., Proc Natl Acad Sci USA 85: 2568-72 (1988); DeresiewiczR et al., Biochemistry 31: 3272-80 (1992); Deresiewicz R et al., Mol GenGenet 241: 467-73 (1993); Ohmura M et al., Microb Pathog 15: 169-76(1993); Cao C et al., Microbiol Immunol 38: 441-7 (1994); Suhan M, HovdeC, Infect Immun 66: 5252-9 (1998)). Mutating both glutamate-167 andarginine-170 eliminated the enzymatic activity of Slt-I A1 in acell-free ribosome inactivation assay (LaPointe, J Biol Chem 280:23310-18 (2005)). In another approach using de novo expression of Slt-IA1 in the endoplasmic reticulum, mutating both glutamate-167 andarginine-170 eliminated Slt-I A1 fragment cytotoxicity at thatexpression level (LaPointe, J Biol Chem 280: 23310-18 (2005)). Atruncation analysis demonstrated that a fragment of StxA from residues75 to 268 still retains significant enzymatic activity in vitro (Haddad,J Bacteriol 175: 4970-8 (1993)). A truncated fragment of Slt-I A1containing residues 1-239 displayed significant enzymatic activity invitro and cytotoxicity by de novo expression in the cytosol (LaPointe, JBiol Chem 280: 23310-18 (2005)). Expression of a Slt-I A1 fragmenttruncated to residues 1-239 in the endoplasmic reticulum was notcytotoxic because it could not retrotranslocate into the cytosol(LaPointe, J Biol Chem 280: 23310-18 (2005)).

For the purposes of the present invention, the specific order ororientation is not fixed for the Shiga toxin effector region and theCD20 binding region in relation to each other or the entire CD20-bindingprotein's N-terminal(s) and C-terminal(s) (see e.g. FIG. 1). In theabove CD20-binding proteins, the CD20 binding regions and Shiga toxineffector regions may be directly linked to each other and/or suitablylinked to each other via one or more intervening polypeptide sequences,such as with one or more linkers well known in the art.

II. Examples of Specific Structural Variations of the CD20-BindingProteins of the Invention

Among certain embodiments of the present invention, the CD20-bindingproteins comprise the Shiga toxin effector region comprising orconsisting essentially of amino acids 75 to 251 of SLT-1A (SEQ ID NO:1),StxA (SEQ ID NO:25), or SLT-2A (SEQ ID NO:26). Further embodiments areCD20-binding proteins in which the Shiga toxin effector region comprisesor consists essentially of amino acids 1 to 241 of SLT-1A (SEQ ID NO:1),StxA (SEQ ID NO:25), and/or SLT-2A (SEQ ID NO:26). Further embodimentsare CD20-binding proteins in which the Shiga toxin effector regioncomprises or consists essentially of amino acids 1 to 251 of SLT-1A (SEQID NO:1), StxA (SEQ ID NO:25), and/or SLT-2A (SEQ ID NO:26). Furtherembodiments are CD20-binding proteins in which the Shiga toxin effectorregion comprises or consists essentially of amino acids 1 to 261 ofSLT-1A (SEQ ID NO:1), StxA (SEQ ID NO:25), and/or SLT-2A (SEQ ID NO:26).

For certain embodiments, the CD20-binding proteins of the presentinvention is one which comprises or consists essentially of the aminoacid sequence of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:14, or SEQ IDNO:16.

As used herein, the term “heavy chain variable (V_(H)) domain” or “lightchain variable (V_(L)) domain” respectively refer to any antibody V_(H)or V_(L) domain (e.g. a human V_(H) or V_(L) domain) as well as anyderivative thereof retaining at least qualitative antigen bindingability of the corresponding native antibody (e.g. a humanized V_(H) orV_(L) domain derived from a native murine V_(H) or V_(L) domain). AV_(H) or V_(L) domain consists of a “framework” region interrupted bythe three CDRs. The framework regions serve to align the CDRs forspecific binding to an epitope of an antigen. From amino-terminus tocarboxyl-terminus, both V_(H) and V_(L) domains comprise the followingframework (FR) and CDR regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, andFR4. The assignment of amino acids to each domain is in accordance withthe definitions of Kunik V et al., PLoS Comput Biol 8: e1002388 (2012)and Kunik V et al., Nucleic Acids Res 40: W521-4 (2012) or alternativelyin accordance with the definitions of Kabat, Sequences of Proteins ofImmunological Interest, (5th ed., National Institutes of Health,Bethesda, Md., 1991); or Chothia and Lesk, J. Mol. Biol. 196: 901-17(1987); Chothia et al., Nature 342: 878-83 (1989).

In certain embodiments of the invention, the CDRs comprise three CDRsderived from a heavy chain of the antibody and three CDRs derived from alight chain of the antibody. In certain embodiments, the three heavychain CDRs comprise SEQ ID NO: 7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQID NO:9 (HCR3), while the three light chain CDRs comprise SEQ ID NO:9(LCDR1), SEQ ID NO:10 (LCDR2) and SEQ ID NO:11 (LCDR3). Additionally, incertain embodiments of the invention, the immunoglobulin-type bindingregion comprises or consists essentially of amino acids 2 to 245 of SEQID NO:4.

It is within the scope of the invention to use fragments, variants,and/or derivatives of the polypeptides of the CD20-binding proteins ofthe invention which contain a functional CD20 binding site to anyextracellular part of CD20, and even more preferably capable of bindingCD20 with high affinity (e.g. as shown by K_(D)). For example, theinvention provides immunoglobulin-derived polypeptide sequences that canbind to CD20. Any polypeptide may be substituted for this region whichbinds an extracellular part of CD20 with a dissociation constant (K_(D))of 10⁻⁵ to 10⁻¹² moles/liter, preferably less than 200 nM.

Thus it is within the scope of the invention to alter theimmunoglobulin-type binding site of a disclosed exemplary CD20-bindingprotein so long as at least one polypeptide sequence is chosen from thegroup consisting of the CDR1 sequences, CDR2 sequences, and CDR3sequences that are described. In particular, but without limitation, thepolypeptide sequences of the invention may consist essentially of 4framework regions (FR1 to FR4) and three complementary determiningregions (CDR1 to CDR3 respectively); or any suitable fragment of suchamino acid sequence that exhibits target biomolecule bindingfunctionality based on the presence of one or more CDRs.

In certain embodiments, the immunoglobulin-type binding region comprises(i) a heavy chain variable (VH) domain comprising CDR amino acidsequences as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, and(ii) a light chain variable (VL) domain comprising CDR amino acidsequences as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11. Inother embodiments, the immunoglobulin-type binding region comprises orconsists essentially of amino acids 2 to 245 of SEQ ID NO:4.

Among certain embodiments of the present invention, theimmunoglobulin-type binding region is derived from a nanobody or singledomain immunoglobulin-derived region V_(HH) which exhibits high affinitybinding specifically to CD20. Generally, nanobodies are constructed fromfragments of naturally occurring single, monomeric variable domainantibodies (sdAbs) of the sort found in camelids and cartilaginousfishes (Chondrichthyes). Nanobodies are engineered from these naturallyoccurring antibodies by truncating the single, monomeric variable domainto create a smaller and more stable molecule. Due to their small size,nanobodies are able to bind to antigens that are not accessible to wholeantibodies.

III. The General Function of the CD20-Binding Proteins of the Invention

The present invention provides various CD20-binding proteins for theselective killing of specific cell types, the CD20 proteinscomprising 1) immunoglobulin-type CD20 binding regions for celltargeting and 2) cytotoxic Shiga toxin effector regions for inducingcellular internalization and, optionally, cell killing as well. Thelinking of CD20 targeting immunoglobulin-type binding regions withShiga-toxin-Subunit-A-derived regions enables the targeting of thepotent Shiga toxin cytotoxicity specifically to CD20 expressing cells.In their preferred embodiments, the CD20-binding proteins of theinvention are capable of binding CD20 natively present on a cell surfaceand entering the cell. Once internalized within a targeted cell type,certain embodiments of the CD20-binding proteins of the invention arecapable of routing a cytotoxic Shiga toxin effector polypeptide fragmentinto the cytosol of the target cell. Once in the cytosol of a targetedcell type, certain embodiments of the CD20-binding proteins of theinvention are capable of enzymatically inactivating ribosomes andeventually killing the cell. Alternatively, non-toxic variants may beused to deliver additional exogenous materials and/or label theinteriors of CD20 expressing cells for diagnostic purposes.

Various types of cells which express CD20 may be targeted by theCD20-binding proteins of the invention for killing and/or receivingexogenous materials. Among the CD20 expressing cell types anticipated tointernalize the CD20-binding proteins of the invention are those withinthe B-cell lineage. “B-cell lineage” is a term used to describe thosecells that are cytologically or otherwise identified as B-cellsthemselves, e.g., through cell surface markers, or were once orpresently derived from cells that are cytologically or otherwiseidentified as B-cells. The term “B-cell lineage” includes neoplasticcells derived from the B-cell lineage or precursors to the B-celllineage. Among the CD20 expressing cell types that may be targeted aredysplastic or neoplastic cells of cell lineages which do not normallyexpress CD20, e.g. melanoma cells. In particular, the CD20 expressingcells to be targeted with the CD20-binding proteins of the inventioninclude neoplastic cells of B-cell lineages or non-B-cell lineages, suchas neoplastic cells from a hematopoietic lineage that are not usuallycategorized as B-cells but which express CD20.

A. CD20-Binding Protein Capable of Inducing Rapid Internalization ofCD20

The Shiga toxin effector regions of the present invention provide aninternalization function, moving the CD20-binding proteins from theexternal surface of the target cell into the cytosol of the target cell.However, this internalization function is also an acceleration functionin that the cellular internalization of CD20 is promoted or induced. Asused in the specification and the claims herein, the phrase “rapidinternalization” refers to a CD20-binding protein of the inventiondecreasing the time for CD20 cellular internalization upon binding ascompared to a prior art reference molecule, such as the monoclonalantibody rituximab.

For the purposes of the present invention, cellular internalization isconsidered rapid if the time for internalization to occur due to thebinding of the CD20-binding proteins is reduced as compared to the timefor internalization of the target molecule with the binding of awell-characterized antibody recognizing a CD20 antigen, such as the 1H4CD20 monoclonal antibody (Haisma H et al., Blood 92: 184-90 (1999)). Forexample, internalization timing for the CD20 antigen, although variablefor cell type and antibody type, does not typically begin to reachmaximal levels until approximately six hours after binding. Thus theterm “rapid” as defined within the present specification is less thanthis six hour standard internalization window. In certain embodiments,rapid can be as quickly as less than about one hour, but can alsoencompass a range of from about 1 hour to about 2 hours, to about 3hours, to about 4 hours, to about 5 hours; a range of about 2 hours toabout 3 hours, to about 4 hours, to about 5 hours; a range of about 3hours to about 4 hours, to about 5 hours; and a range of about 4 hoursto about 5 hours.

B. Cell Kill Via Targeted Shiga Toxin Cytotoxicity

Because members of the Shiga toxin family are adapted to killingeukaryotic cells, CD20-binding proteins designed using Shiga toxineffector regions can show potent cell-kill activity. The A Subunits ofmembers of the Shiga toxin family comprise enzymatic domains capable ofkilling a eukaryotic cell once in the cell's cytosol. Certainembodiments of the CD20-binding proteins of the invention take advantageof this cytotoxic mechanism.

In certain embodiments of the CD20-binding proteins of the invention,upon contacting a cell expressing CD20 such that at least a part of CD20is accessible from the extracellular space, the CD20-binding protein iscapable of causing death of the cell. CD20 positive “cell kill” may beaccomplished using a CD20-binding protein of the invention under variedconditions of target cells, such as an ex vivo manipulated target cell,a target cell cultured in vitro, a target cell within a tissue samplecultured in vitro, or a target cell in vivo.

C. Selective Cytotoxicity Between CD20 Expressing Cells and Non-CD20Expressing Cells

By targeting the delivery of enzymatically active Shiga toxin regionsusing high-affinity immunoglobulin-type binding regions to CD20expressing cells, this potent cell-kill activity can be restricted topreferentially killing CD20 positive cell types.

In certain embodiments, upon administration of the CD20-binding proteinof the invention to a mixture of cell types, the CD20-binding protein iscapable of selectively killing CD20 expressing cells displaying anextracellular CD20 target compared to cell types lacking extracellularCD20 targets. Because members of the Shiga toxin family are adapted forkilling eukaryotic cells, CD20-binding proteins designed using Shigatoxin effector regions can show potent cytotoxic activity. By targetingthe delivery of enzymatically active Shiga toxin regions to CD20expressing cells using high-affinity immunoglobulin-type bindingregions, this potent cell kill activity can be restricted topreferentially killing only CD20 expressing cells.

In certain embodiments, the CD20-binding protein of the invention iscapable of selectively or preferentially causing the death of a specificcell type within a mixture of two or more different cell types. Thisenables targeting cytotoxic activity to specific cell types with a highpreferentiality, such as with at least a 3-fold cytotoxic effect, over“bystander” cell types that do not express any significant amount ofextracellular CD20 targets. This enables the targeted cell-killing ofspecific cell types expressing CD20 on cellular surfaces with a highpreferentiality, such as with at least a 3-fold cytotoxic effect, over“bystander” cell types that do not express significant amounts of CD20or are not exposing significant amounts of CD20 on a cellular surface.

In certain further embodiments, upon administration of the CD20-bindingprotein to two different populations of cell types, the CD20-bindingprotein is capable of causing cell death as defined by the half-maximalcytotoxic concentration (CD₅₀) on a cell population which expresses CD20on a cellular surface at a dose at least three times lower than the CD₅₀dose of the same CD20-binding protein to a cell population which doesnot express CD20.

In certain embodiments, the cytotoxic activity toward populations ofcell types expressing CD20 on a cellular surface is at least 3-foldhigher than the cytotoxic activity toward populations of cell types notphysically coupled with any extracellular CD20 target of the CD20binding region of the embodiment. According to the present invention,selective cytotoxicity may be quantified in terms of the ratio (a/b) of(a) cytotoxicity towards a population of cells expressing anextracellular CD20 target of the CD20 binding region of the embodimentto (b) cytotoxicity towards a population of cells of a cell type notphysically coupled with any extracellular CD20 target of the CD20binding region of the embodiment. In certain embodiments, thecytotoxicity ratio is indicative of selective cytotoxicity which is atleast 3-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold,40-fold, 50-fold, 75-fold, 100-fold, 250-fold, 500-fold, 750-fold, or1000-fold higher for populations of cells or cell types expressing CD20compared to populations of cells or cell types which do not expressCD20.

This preferential cell-killing function allows a targeted cell to bekilled by certain CD20-binding proteins of the invention under variedconditions and in the presence of non-targeted bystander cells, such asex vivo manipulated mixtures of cell types, in vitro cultured tissueswith mixtures of cell types, or in vivo in the presence of multiple celltypes (e.g. in situ or in its native location within a multicellularorganism).

In addition, catalytically inactive forms of CD20-binding proteinsoptionally may be used for diagnostic functions. The conjugating ofadditional diagnostic agents known in the art to CD20-binding proteinsof the invention enable the imaging of intracellular organelles (e.g.Golgi, endoplasmic reticulum, and cytosolic compartments) of individualimmune cells of the B-cell lineage or cancer cells in a patient orbiopsy sample. For example, this may be useful in the diagnosis ofneoplastic cell types, assaying the progression of anticancer therapiesover time, and/or evaluating the presence of residual cancer cells aftersurgical excision of a tumor mass.

D. Delivery of Additional Exogenous Material

Because the CD20-binding protein are capable of inducing cellularinternalization of CD20 after binding to an extracellular part of CD20,certain embodiments of the CD20-binding proteins of the invention may beused to deliver additional exogenous materials into the interior of CD20expressing cells. In one sense, the entire CD20-binding protein is anexogenous material which will enter the cell; thus, the “additional”exogenous materials are materials linked to but other than the coreCD20-binding protein itself.

“Additional exogenous material” as used herein refers to one or moremolecules, often not generally present within a native target cell,where the CD20-binding proteins of the present invention can be used tospecifically transport such material to the interior of a cell. Ingeneral, additional exogenous material is selected from peptides,polypeptides, proteins, and polynucleotides. One example of anadditional exogenous material that is a peptide is an influenza virusantigen, such as the influenza Matrix 58-66 peptide (SEQ ID NO:3). Oneexemplary embodiment of a CD20-binding protein that may deliver thatantigen into a target cell that expresses CD20 is provided in SEQ IDNO:16.

Additional exogenous material may include an interior polypeptidesequence within the core CD20-binding protein structure, such as theinfluenza Matrix 58-66 peptide (SEQ ID NO:3). Similarly, additionalexogenous material may include a terminally-located polypeptide sequencelinked to a terminal of the CD20-binding structure. Certain embodimentsof the CD20-binding proteins of the invention that may deliver thatantigen, as an additional exogenous material, into a target cell thatexpresses CD20 on a cell surface is the CD20-binding protein thatcomprises or consists essentially of SEQ ID NO:4, SEQ ID NO:12, SEQ IDNO:14, or SEQ ID NO:16.

Additional examples of exogenous materials that may be linked to theCD20-binding proteins of the invention include antigens such as thosederived from bacterial proteins, such as those characteristic ofantigen-presenting cells infected by bacteria. Further examples ofadditional exogenous materials are proteins mutated in cancer orproteins that are aberrantly expressed in cancer. Further examples ofadditional exogenous materials include T-cell complementary determiningregions capable of functioning as exogenous antigens.

Further examples of exogenous materials that may be linked to theCD20-binding proteins of the invention include proteins other thanantigens, such as enzymes. Further types of exogenous material arepolynucleotides. Among the polynucleotides that can be transported arethose formulated to have regulatory function, such as small interferingRNA (siRNA) and microRNA (miRNA).

Additional examples of exogenous materials include antigens such asthose derived from bacterial proteins, such as those characteristic ofantigen-presenting cells that are infected with bacteria. Furtherexamples of exogenous antigens are ones that are derived from a proteinmutated in cancer or proteins that are aberrantly expressed in cancer.T-cell complementary determining regions (CDR) can also act as exogenousantigens for the purposes of the present invention. Additional examplesof exogenous material includes proteins other than antigens, such asenzymes. A further type of exogenous material is nucleic acids. Amongthe nucleic acids that can be transported are those formulated to haveregulatory function, such as small interfering RNA (siRNA) and microRNA(miRNA).

Variations in the Polypeptide Sequence of the CD20-Binding Proteins ofthe Invention which Maintain Overall Structure and Function

In certain of the above embodiments, the CD20-binding protein of theinvention is a variant in which there are one or more conservative aminoacid substitutions introduced into the polypeptide region(s). As usedherein, the term “conservative substitution” denotes that one or moreamino acids are replaced by another, biologically similar amino acidresidue. Examples include substitution of amino acid residues withsimilar characteristics, e.g. small amino acids, acidic amino acids,polar amino acids, basic amino acids, hydrophobic amino acids andaromatic amino acids (see, for example, Table B below). An example of aconservative substitution with a residue normally not found inendogenous, mammalian peptides and proteins is the conservativesubstitution of an arginine or lysine residue with, for example,ornithine, canavanine, aminoethylcysteine, or another basic amino acid.For further information concerning phenotypically silent substitutionsin peptides and proteins (see e.g. Bowie J et al., Science 247: 1306-10(1990)). In the scheme below are conservative substitutions of aminoacids grouped by physicochemical properties. I: neutral, hydrophilic,II: acids and amides, III: basic, IV: hydrophobic, V: aromatic, bulkyamino acids.

TABLE B Examples of Conservative Amino Acid Substitutions I II III IV VA N H M F S D R L Y T E K I W P Q V G C

In certain embodiments, a CD20-binding protein of the invention maycomprise functional fragments or variants of a polypeptide region of theinvention that have, at most, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1amino acid substitutions compared to a polypeptide sequence recitedherein, as long as it retains measurable biological activity alone or asa component of a CD20-binding protein. Variants of CD20-binding proteinsare within the scope of the invention as a result of changing apolypeptide of the CD20-binding protein by altering one or more aminoacids or deleting or inserting one or more amino acids, such as withinthe immunoglobulin-type binding region or the Shiga toxin effectorregion, in order to achieve desired properties, such as changedcytotoxicity, changed cytostatic effects, changed immunogenicity, and/orchanged serum half-life. A polypeptide of a CD20-binding protein of theinvention may further be with or without a signal sequence.

In certain embodiments, a CD20-binding protein of the invention sharesat least 85%, 90%, 95%, 96%, 97%, 98%, 99% or more amino acid sequenceidentity to any one of the amino acid sequences of a CD20-bindingprotein recited herein, as long as it retains measurable biologicalactivity, such as cytotoxicity, extracellular target biomoleculebinding, enzymatic catalysis, or subcellular routing. Theimmunoglobulin-type binding region may differ from the amino acidsequences of a CD20-binding protein recited herein, as long as itretains binding functionality to its extracellular target biomolecule.Binding functionality will most likely be retained if the amino acidsequences of the ABRs are identical. For example, a CD20-binding proteinthat consists essentially of 85% amino acid identity to SEQ ID NO: 4 orSEQ II) NO:16 in which for the purposes of determining the degree ofamino acid identity, the amino acid residues that form the ABR aredisregarded. Binding functionality can be determined by the skilledworker using standard techniques.

In certain embodiments, the Shiga toxin effector region may be alteredto change the enzymatic activity and/or cytotoxicity of the Shiga toxineffector region. This change may or may not result in a change in thecytotoxicity of a CD20-binding protein of which the altered Shiga toxineffector region is a component. Possible alterations include mutationsto the Shiga toxin effector region selected from the group consistingof: a truncation, deletion, inversion, insertion and substitution.

The cytotoxicity of the A Subunits of members of the Shiga toxin familymay be reduced or eliminated by mutation or truncation. The positionslabeled tyrosine-77, glutamate-167, arginine-170, tyrosine-114, andtryptophan-203 have been shown to be important for the catalyticactivity of Stx, Stx1, and Stx2 (Hovde C et al., Proc Natl Acad Sci USA85: 2568-72 (1988); Deresiewicz R et al., Biochemistry 31: 3272-80(1992); Deresiewicz R et al., Mol Gen Genet 241: 467-73 (1993); Ohmura Met al., Microb Pathog 15: 169-76 (1993); Cao C et al., Microbiol Immunol38: 441-7 (1994); Suhan M, Hovde C, Infect Immun 66: 5252-9 (1998)).Mutating both glutamate-167 and arginine-170 eliminated the enzymaticactivity of Slt-I A1 in a cell-free ribosome inactivation assay(LaPointe, J Biol Chem 280: 23310-18 (2005)). In another approach usingde novo expression of Slt-I A1 in the endoplasmic reticulum, mutatingboth glutamate-167 and arginine-170 eliminated Slt-I A1 fragmentcytotoxicity at that expression level (LaPointe, J Biol Chem 280:23310-18 (2005)). A truncation analysis demonstrated that a fragment ofStxA from residues 75 to 268 still retains significant enzymaticactivity in vitro (Haddad, J Bacteriol 175: 4970-8 (1993)). A truncatedfragment of Slt-I A1 containing residues 1-239 displayed significantenzymatic activity in vitro and cytotoxicity by de novo expression inthe cytosol (LaPointe, J Biol Chem 280: 23310-18 (2005)). Expression ofa Slt-I A1 fragment truncated to residues 1-239 in the endoplasmicreticulum was not cytotoxic because it could not retrotranslocate to thecytosol (LaPointe, J Biol Chem 280: 23310-18 (2005)).

The most critical residues for enzymatic activity and/or cytotoxicity inthe Shiga toxin A Subunits were mapped to the followingresidue-positions: aspargine-75, tyrosine-77, glutamate-167,arginine-170, and arginine-176 among others (Di, Toxicon 57: 535-39(2011)). In particular, a double-mutant construct of Stx2A containingglutamate-E167-to-lysine and arginine-176-to-lysine mutations wascompletely inactivated; whereas, many single mutations in Stx1 and Stx2showed a 10-fold reduction in cytotoxicity. Further, truncation of Stx1Ato 1-239 or 1-240 reduced its cytotoxicity, and similarly, truncation ofStx2A to a conserved hydrophobic residue reduced its cytotoxicity.

Shiga-like toxin 1 A Subunit truncations are catalytically active,capable of enzymatically inactivating ribosomes in vitro, and cytotoxicwhen expressed within a cell (LaPointe, J Biol Chem 280: 23310-18(2005)). The smallest Shiga toxin A Subunit fragment exhibiting fullenzymatic activity is a polypeptide composed of residues 1-239 of Slt1A(LaPointe, J Biol Chem 280: 23310-18 (2005)). Although the smallestfragment of the Shiga toxin A Subunit reported to retain substantialcatalytic activity was residues 75-247 of StxA (Al-Jaufy, Infect Immun62: 956-60 (1994)), a StxA truncation expressed de novo within aeukaryotic cell requires only up to residue 240 to reach the cytosol andexert catalytic inactivation of ribosomes (LaPointe, J Biol Chem 280:23310-18 (2005)).

In certain embodiments derived from SLT-1A (SEQ ID NO:1), StxA (SEQ IDNO:25), or SLT-2A (SEQ ID NO:26), these changes include substitution ofthe asparagine at position 75, tyrosine at position 77, tyrosine atposition 114, glutamate at position 167, arginine at position 170,arginine at position 176, and/or substitution of the tryptophan atposition 203. Examples of such substitutions will be known to theskilled worker based on the prior art, such as asparagine at position 75to alanine, tyrosine at position 77 to serine, substitution of thetyrosine at position 114 to alanine, substitution of the glutamate atposition 167 to aspartate, substitution of the arginine at position 170to alanine, substitution of the arginine at position 176 to lysine,and/or substitution of the tryptophan at position 203 to alanine.

CD20-binding proteins of the invention may optionally be conjugated toone or more additional agents which may include therapeutic and/ordiagnostic agents known in the art.

Production, Manufacture, and Purification of a CD20-Binding Protein ofthe Invention

The CD20-binding proteins of the invention may be produced usingbiochemical engineering techniques well known to those of skill in theart. For example, CD20-binding proteins of the invention may bemanufactured by standard synthetic methods, by use of recombinantexpression systems, or by any other suitable method. Thus, theCD20-binding proteins may be synthesized in a number of ways, including,e.g. methods comprising: (1) synthesizing a polypeptide or polypeptidecomponent of a CD20-binding protein using standard solid-phase orliquid-phase methodology, either stepwise or by fragment assembly, andisolating and purifying the final peptide compound product; (2)expressing a polynucleotide that encodes a polypeptide or polypeptidecomponent of a CD20-binding protein in a host cell and recovering theexpression product from the host cell or host cell culture; or (3)cell-free in vitro expression of a polynucleotide encoding a polypeptideor polypeptide component of a CD20-binding protein, and recovering theexpression product; or by any combination of the methods of (1), (2) or(3) to obtain fragments of the peptide component, subsequently joining(e.g. ligating) the fragments to obtain the peptide component, andrecovering the peptide component.

It may be preferable to synthesize a polypeptide or polypeptidecomponent of a CD20-binding protein of the invention by means ofsolid-phase or liquid-phase peptide synthesis. CD20-binding proteins ofthe invention may suitably be manufactured by standard syntheticmethods. Thus, peptides may be synthesized by, e.g. methods comprisingsynthesizing the peptide by standard solid-phase or liquid-phasemethodology, either stepwise or by fragment assembly, and isolating andpurifying the final peptide product. In this context, reference may bemade to WO 1998/11125 or, inter alia, Fields, G et al., Principles andPractice of Solid-Phase Peptide Synthesis (Synthetic Peptides, GregoryA. Grant, ed., Oxford University Press, U.K., 2nd ed., 2002) and thesynthesis examples therein.

CD20-binding proteins of the invention may be prepared (produced andpurified) using recombinant techniques well known in the art. Ingeneral, methods for preparing polypeptides by culturing host cellstransformed or transfected with a vector comprising the encodingpolynucleotide and recovering the polypeptide from cell culture aredescribed in, e.g. Sambrook et al., Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Laboratory Press, NY, U.S., 1989);Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring HarborLaboratory Press, N.Y., U.S., 1995). Any suitable host cell may be usedto produce a CD20-binding protein of the invention. Host cells may becells stably or transiently transfected, transformed, transduced orinfected with one or more expression vectors which drive expression of apolypeptide of the invention. In addition, a CD20-binding protein of theinvention may be produced by modifying the polynucleotide encoding theCD20-binding protein that result in altering one or more amino acids ordeleting or inserting one or more amino acids in order to achievedesired properties, such as changed cytotoxicity, changed cytostaticeffects, changed immunogenicity, and/or changed serum half-life.

Accordingly, the present invention also provides methods for producing aCD20-binding protein of the invention according to above recited methodsand using a polynucleotide encoding part or all of a polypeptide of theinvention, an expression vector comprising at least one polynucleotideof the invention capable of encoding part or all of a polypeptide of theinvention when introduced into a host cell, and/or a host cellcomprising a polynucleotide or expression vector of the invention.

When a polypeptide or protein is expressed using recombinant techniquesin a host cell or cell-free system, it is advantageous to separate (orpurify) the desired polypeptide or protein away from other components,such as host cell factors, in order to obtain preparations that are ofhigh purity or are substantially homogeneous. Purification can beaccomplished by methods well known in the art, such as centrifugationtechniques, extraction techniques, chromatographic and fractionationtechniques (e.g. size separation by gel filtration, charge separation byion-exchange column, hydrophobic interaction chromatography, reversephase chromatography, chromatography on silica or cation-exchange resinssuch as DEAE and the like, chromatofocusing, and Protein A Sepharosechromatography to remove contaminants), and precipitation techniques(e.g. ethanol precipitation or ammonium sulfate precipitation. Anynumber of biochemical purification techniques may be used to increasethe purity of a CD20-binding protein of the invention. In certainembodiments, the CD20-binding proteins of the invention may optionallybe purified in homo-multimeric forms (i.e. a protein complex of two ormore identical CD20-binding proteins).

In the Examples below are descriptions of non-limiting examples ofmethods for producing a CD20-binding protein of the invention, as wellas specific but non-limiting aspects of CD20-binding protein productionfor the disclosed, exemplary, CD20-binding proteins.

Pharmaceutical Compositions Comprising a CD20-Binding Protein of theInvention

The present invention provides CD20-binding proteins for use, alone orin combination with one or more additional therapeutic agents, in apharmaceutical composition, for treatment or prophylaxis of conditions,diseases, disorders, or symptoms described in further detail below (e.g.cancers, malignant tumors, non-malignant tumors, and immune disorders).The present invention further provides pharmaceutical compositionscomprising a CD20-binding protein of the invention, or apharmaceutically acceptable salt or solvate thereof, according to theinvention, together with at least one pharmaceutically acceptablecarrier, excipient, or vehicle. In certain embodiments, thepharmaceutical composition of the invention may comprise homo-multimericand/or hetero-multimeric forms of the CD20-binding proteins of theinvention. The pharmaceutical compositions will be useful in methods oftreating, ameliorating, or preventing a disease, condition, disorder, orsymptom described in further detail below. Each such disease, condition,disorder, or symptom is envisioned to be a separate embodiment withrespect to uses of a pharmaceutical composition according to theinvention. The invention further provides pharmaceutical compositionsfor use in at least one method of treatment according to the invention,as described in more detail below.

As used herein, the terms “patient” and “subject” are usedinterchangeably to refer to any organism, commonly vertebrates such ashumans and animals, which presents symptoms, signs, and/or indicationsof at least one disease, disorder, or condition. These terms includemammals such as the non-limiting examples of primates, livestock animals(e.g. cattle, horses, pigs, sheep, goats, etc.), companion animals (e.g.cats, dogs, etc.) and laboratory animals (e.g. mice, rabbits, rats,etc.).

As used herein, “treat,” “treating,” or “treatment” and grammaticalvariants thereof refer to an approach for obtaining beneficial ordesired clinical results. The terms may refer to slowing the onset orrate of development of a condition, disorder or disease, reducing oralleviating symptoms associated with it, generating a complete orpartial regression of the condition, or some combination of any of theabove. For the purposes of this invention, beneficial or desiredclinical results include, but are not limited to, reduction oralleviation of symptoms, diminishment of extent of disease,stabilization (e.g. not worsening) of state of disease, delay or slowingof disease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treat,” “treating,” or “treatment” can also meanprolonging survival relative to expected survival time if not receivingtreatment. A subject (e.g. a human) in need of treatment may thus be asubject already afflicted with the disease or disorder in question. Theterms “treat,” “treating,” or “treatment” includes inhibition orreduction of an increase in severity of a pathological state or symptomsrelative to the absence of treatment, and is not necessarily meant toimply complete cessation of the relevant disease, disorder, orcondition.

As used herein, the terms “prevent,” “preventing,” “prevention” andgrammatical variants thereof refer to an approach for preventing thedevelopment of, or altering the pathology of, a condition, disease, ordisorder. Accordingly, “prevention” may refer to prophylactic orpreventive measures. For the purposes of this invention, beneficial ordesired clinical results include, but are not limited to, prevention orslowing of symptoms, progression or development of a disease, whetherdetectable or undetectable. A subject (e.g. a human) in need ofprevention may thus be a subject not yet afflicted with the disease ordisorder in question. The term “prevention” includes slowing the onsetof disease relative to the absence of treatment, and is not necessarilymeant to imply permanent prevention of the relevant disease, disorder orcondition. Thus “preventing” or “prevention” of a condition may incertain contexts refer to reducing the risk of developing the condition,or preventing or delaying the development of symptoms associated withthe condition.

As used herein, an “effective amount” or “therapeutically effectiveamount” is an amount or dose of a composition (e.g. a therapeuticcomposition or agent) that produces at least one desired therapeuticeffect in a subject, such as preventing or treating a target conditionor beneficially alleviating a symptom associated with the condition. Themost desirable therapeutically effective amount is an amount that willproduce a desired efficacy of a particular treatment selected by one ofskill in the art for a given subject in need thereof. This amount willvary depending upon a variety of factors understood by the skilledworker, including but not limited to the characteristics of thetherapeutic compound (including activity, pharmacokinetics,pharmacodynamics, and bioavailability), the physiological condition ofthe subject (including age, sex, disease type, disease stage, generalphysical condition, responsiveness to a given dosage, and type ofmedication), the nature of the pharmaceutically acceptable carrier orcarriers in the formulation, and the route of administration. Oneskilled in the clinical and pharmacological arts will be able todetermine a therapeutically effective amount through routineexperimentation, namely by monitoring a subject's response toadministration of a compound and adjusting the dosage accordingly (seee.g. Remington: The Science and Practice of Pharmacy (Gennaro A, ed.,Mack Publishing Co., Easton, Pa., U.S., 19th ed., 1995)).

Production or Manufacture of a Pharmaceutical Composition Comprising aCD20-Binding Protein of the Invention

Pharmaceutically acceptable salts or solvates of any of the CD20-bindingproteins of the invention are likewise within the scope of the presentinvention.

The term “solvate” in the context of the present invention refers to acomplex of defined stoichiometry formed between a solute (in casu, apolypeptide compound or pharmaceutically acceptable salt thereofaccording to the invention) and a solvent. The solvent in thisconnection may, for example, be water, ethanol or anotherpharmaceutically acceptable, typically small-molecular organic species,such as, but not limited to, acetic acid or lactic acid. When thesolvent in question is water, such a solvate is normally referred to asa hydrate.

CD20-binding proteins of the present invention, or salts thereof, may beformulated as pharmaceutical compositions prepared for storage oradministration, which typically comprise a therapeutically effectiveamount of a compound of the invention, or a salt thereof, in apharmaceutically acceptable carrier. The term “pharmaceuticallyacceptable carrier” includes any of the standard pharmaceuticalcarriers. Pharmaceutically acceptable carriers for therapeutic use arewell known in the pharmaceutical art, and are described, for example, inRemington's Pharmaceutical Sciences (Mack Publishing Co. (A. Gennaro,ed., 1985)). As used herein, “pharmaceutically acceptable carrier”includes any and all physiologically acceptable, i.e. compatible,solvents, dispersion media, coatings, antimicrobial agents, isotonic,and absorption delaying agents, and the like. Pharmaceuticallyacceptable carriers or diluents include those used in formulationssuitable for oral, rectal, nasal or parenteral (including subcutaneous,intramuscular, intravenous, intradermal, and transdermal)administration. Exemplary pharmaceutically acceptable carriers includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. Examples of suitable aqueous and nonaqueous carriers thatmay be employed in the pharmaceutical compositions of the inventioninclude water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyloleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants. In certain embodiments, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g. by injection or infusion). Depending onselected route of administration, the CD20-binding protein or otherpharmaceutical component may be coated in a material intended to protectthe compound from the action of low pH and other natural inactivatingconditions to which the active CD20-binding protein may encounter whenadministered to a patient by a particular route of administration.

The formulations of the pharmaceutical compositions of the invention mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy. In such form, thecomposition is divided into unit doses containing appropriate quantitiesof the active component. The unit dosage form can be a packagedpreparation, the package containing discrete quantities of thepreparations, for example, packeted tablets, capsules, and powders invials or ampoules. The unit dosage form can also be a capsule, cachet,or tablet itself, or it can be the appropriate number of any of thesepackaged forms. It may be provided in single dose injectable form, forexample in the form of a pen. Compositions may be formulated for anysuitable route and means of administration. Subcutaneous or transdermalmodes of administration may be particularly suitable for therapeuticCD20-binding proteins described herein.

The pharmaceutical compositions of the invention may also containadjuvants such as preservatives, wetting agents, emulsifying agents anddispersing agents. Prevention of the presence of microorganisms may beensured both by sterilization procedures, and by the inclusion ofvarious antibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. Isotonic agents, suchas sugars, sodium chloride, and the like into the compositions, may alsobe desirable. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as, aluminum monostearate and gelatin.

A pharmaceutical composition of the invention also optionally includes apharmaceutically acceptable antioxidant. Exemplary pharmaceuticallyacceptable antioxidants are water soluble antioxidants such as ascorbicacid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,sodium sulfite and the like; oil-soluble antioxidants, such as ascorbylpalmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene(BHT), lecithin, propylgallate, alpha-tocopherol, and the like; andmetal chelating agents, such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

In another aspect, the present invention provides pharmaceuticalcompositions comprising one or a combination of different CD20-bindingproteins of the invention, or an ester, salt or amide of any of theforegoing, and at least one pharmaceutically acceptable carrier.

Therapeutic compositions are typically sterile and stable under theconditions of manufacture and storage. The composition may be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier may be a solvent ordispersion medium containing, for example, water, alcohol such asethanol, polyol (e.g. glycerol, propylene glycol, and liquidpolyethylene glycol), or any suitable mixtures. The proper fluidity maybe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by use of surfactants according to formulation chemistry well knownin the art. In certain embodiments, isotonic agents, e.g. sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride may bedesirable in the composition. Prolonged absorption of injectablecompositions may be brought about by including in the composition anagent that delays absorption for example, monostearate salts andgelatin.

Solutions or suspensions used for intradermal or subcutaneousapplication typically include one or more of: a sterile diluent such aswater for injection, saline solution, fixed oils, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents; antibacterialagents such as benzyl alcohol or methyl parabens; antioxidants such asascorbic acid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates; and tonicity adjusting agents such as, e.g., sodium chlorideor dextrose. The pH can be adjusted with acids or bases, such ashydrochloric acid or sodium hydroxide, or buffers with citrate,phosphate, acetate and the like. Such preparations may be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic.

Sterile injectable solutions may be prepared by incorporating aCD20-binding protein of the invention in the required amount in anappropriate solvent with one or a combination of ingredients describedabove, as required, followed by sterilization microfiltration.Dispersions may be prepared by incorporating the active compound into asterile vehicle that contains a dispersion medium and other ingredients,such as those described above. In the case of sterile powders for thepreparation of sterile injectable solutions, the methods of preparationare vacuum drying and freeze-drying (lyophilization) that yield a powderof the active ingredient in addition to any additional desiredingredient from a sterile-filtered solution thereof.

When a therapeutically effective amount of a CD20-binding protein of theinvention is designed to be administered by, e.g. intravenous, cutaneousor subcutaneous injection, the binding agent will be in the form of apyrogen-free, parenterally acceptable aqueous solution. Methods forpreparing parenterally acceptable protein solutions, taking intoconsideration appropriate pH, isotonicity, stability, and the like, arewithin the skill in the art. A preferred pharmaceutical composition forintravenous, cutaneous, or subcutaneous injection will contain, inaddition to binding agents, an isotonic vehicle such as sodium chlorideinjection, Ringer's injection, dextrose injection, dextrose and sodiumchloride injection, lactated Ringer's injection, or other vehicle asknown in the art. A pharmaceutical composition of the present inventionmay also contain stabilizers, preservatives, buffers, antioxidants, orother additives well known to those of skill in the art.

As described elsewhere herein, a compound may be prepared with carriersthat will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art (see e.g. Sustained andControlled Release Drug Delivery Systems (J. Robinson, ed., MarcelDekker, Inc., NY, U.S., 1978)).

In certain embodiments, the pharmaceutical composition of the inventionmay be formulated to ensure a desired distribution in vivo. For example,the blood-brain barrier excludes many large and/or hydrophiliccompounds. To target a therapeutic compound or composition of theinvention to a particular in vivo location, they can be formulated, forexample, in liposomes which may comprise one or more moieties that areselectively transported into specific cells or organs, thus enhancingtargeted drug delivery. Exemplary targeting moieties include folate orbiotin; mannosides; antibodies; surfactant protein A receptor; p120catenin and the like.

Polynucleotides, Expression Vectors, and Host Cells

Beyond the CD20-binding proteins of the present invention, thepolynucleotides which encode such CD20-binding proteins, or functionalportions thereof, are within the scope of the present invention. Theterm “polynucleotide” is equivalent to the term “nucleic acids” both ofwhich include polymers of deoxyribonucleic acids (DNAs), polymers ofribonucleic acids (RNAs), analogs of these DNAs or RNAs generated usingnucleotide analogs, and derivatives, fragments and homologs thereof. Thepolynucleotide of the invention may be single-, double-, ortriple-stranded. Disclosed polynucleotides are specifically disclosed toinclude all polynucleotides capable of encoding an exemplaryCD20-binding protein, for example, taking into account the wobble knownto be tolerated in the third position of RNA codons, yet encoding forthe same amino acid as a different RNA codon (see Stothard P,Biotechniques 28: 1102-4 (2000)).

In one aspect, the invention provides polynucleotides which encode aCD20-binding protein of the invention, or a fragment or derivativethereof. The polynucleotides may include, e.g., nucleic acid sequenceencoding a polypeptide at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 99% or more, identical to a polypeptide comprising one of theamino acid sequences of the CD20-binding protein. The invention alsoincludes polynucleotides comprising nucleotide sequences that hybridizeunder stringent conditions to a polynucleotide which encodes aCD20-binding protein of the invention, or a fragment or derivativethereof, or the antisense or complement of any such sequence.

Derivatives or analogs of the polynucleotides (or CD20-binding proteins)of the invention include, inter alia, polynucleotide (or polypeptide)molecules having regions that are substantially homologous to thepolynucleotides or CD20-binding proteins of the invention, e.g. by atleast about 45%, 50%, 70%, 80%, 95%, 98%, or even 99% identity (with apreferred identity of 80-99%) over a polynucleotide or polypeptidesequence of the same size or when compared to an aligned sequence inwhich the alignment is done by a computer homology program known in theart. An exemplary program is the GAP program (Wisconsin SequenceAnalysis Package, Version 8 for UNIX, Genetics Computer Group,University Research Park, Madison, Wis., U.S.) using the defaultsettings, which uses the algorithm of Smith T, Waterman M, Adv. Appl.Math. 2: 482-9 (1981). Also included are polynucleotides capable ofhybridizing to the complement of a sequence encoding the proteins of theinvention under stringent conditions (see e.g. Ausubel F, et al.,Current Protocols in Molecular Biology (John Wiley & Sons, New York,N.Y., U.S., 1993)), and below. Stringent conditions are known to thoseskilled in the art and may be found in Current Protocols in MolecularBiology (John Wiley & Sons, NY, U.S., Ch. Sec. 6.3.1-6.3.6 (1989).

Further, the present invention further provides expression vectors thatcomprise the polynucleotides within the scope of the invention. Thepolynucleotides capable of encoding the CD20-binding proteins of theinvention may be inserted into known vectors, including bacterialplasmids, viral vectors and phage vectors, using material and methodswell known in the art to produce expression vectors. Such expressionvectors will include the polynucleotides necessary to support productionof contemplated CD20-binding proteins within any host cell of choice orcell-free expression systems (e.g. pTxb1 and pIVEX2.3 described in theExamples below). The specific polynucleotides comprising expressionvectors for use with specific types of host cells or cell-freeexpression systems are well known to one of ordinary skill in the art,can be determined using routine experimentation, or may be purchased.

The term “expression vector,” as used herein, refers to apolynucleotide, linear or circular, comprising one or more expressionunits. The term “expression unit” denotes a polynucleotide segmentencoding a polypeptide of interest and capable of providing expressionof the nucleic acid segment in a host cell. An expression unit typicallycomprises a transcription promoter, an open reading frame encoding thepolypeptide of interest, and a transcription terminator, all in operableconfiguration. An expression vector contains one or more expressionunits. Thus, in the context of the present invention, an expressionvector encoding a CD20-binding protein comprising a single polypeptidechain (e.g. an scFv linked to a Shiga toxin effector region) includes atleast an expression unit for the single polypeptide chain, whereas aCD20-binding protein comprising, e.g. two or more polypeptide chains(e.g. one chain comprising a V_(L) domain and a second chain comprisinga V_(H) domain linked to a toxin effector region) includes at least twoexpression units, one for each of the two polypeptide chains of theCD20-binding protein. For expression of multi-chain CD20-bindingproteins, an expression unit for each polypeptide chain may also beseparately contained on different expression vectors (e.g. expressionmay be achieved with a single host cell into which expression vectorsfor each polypeptide chain has been introduced).

Expression vectors capable of directing transient or stable expressionof polypeptides and proteins are well known in the art. The expressionvectors generally include, but are not limited to, one or more of thefollowing: a heterologous signal sequence or peptide, an origin ofreplication, one or more marker genes, an enhancer element, a promoter,and a transcription termination sequence, each of which is well known inthe art. Optional regulatory control sequences, integration sequences,and useful markers that can be employed are known in the art.

The term “host cell” refers to a cell which can support the replicationor expression of the expression vector. Host cells may be prokaryoticcells, such as E. coli or eukaryotic cells (e.g. yeast, insect,amphibian, bird, or mammalian cells). Creation and isolation of hostcell lines comprising a polynucleotide of the invention or capable ofproducing a CD20-binding protein of the invention can be accomplishedusing standard techniques known in the art.

CD20-binding proteins within the scope of the present invention may bevariants or derivatives of the CD20-binding proteins described hereinthat are produced by modifying the polynucleotide encoding aCD20-binding protein by altering one or more amino acids or deleting orinserting one or more amino acids that may render it more suitable toachieve desired properties, such as more optimal expression by a hostcell.

Methods for Using a CD20-Binding Protein or a Pharmaceutical Compositionof the Invention

Generally, it is an object of the invention to provide pharmacologicallyactive agents, as well as compositions comprising the same, that can beused in the prevention and/or treatment of diseases, disorders, andconditions, such as cancers, tumors, immune disorders, or furtherpathological conditions mentioned herein. Accordingly, the presentinvention provides methods of using the CD20-binding proteins andpharmaceutical compositions of the invention for the killing of CD20expressing cells, delivering of additional exogenous materials into CD20expressing cells, labeling of the interior of CD20 expressing cells, andfor treating diseases, disorders, and conditions as described herein.

In particular, it is an object of the invention to provide suchpharmacologically active agents, compositions, and/or methods that havecertain advantages compared to the agents, compositions, and/or methodsthat are currently known in the art. Accordingly, the present inventionprovides methods of using CD20-binding proteins with specifiedpolypeptide sequences and pharmaceutical compositions thereof. Forexample, any of the polypeptide sequences in SEQ ID NOs:1, 3, 4, 6-12,14, 16, and/or 18-29, can be specifically utilized as a component of theCD20-binding protein used in the following methods.

The present invention provides methods of killing a CD20 expressing cellcomprising the step of contacting the cell, either in vitro or in vivo,with a CD20-binding protein or pharmaceutical composition of the presentinvention. In certain embodiments, a CD20-binding protein orpharmaceutical composition of the present invention can be used to killCD20 expressing cells in a mixture of different cell types includingnon-CD20 expressing cells, such as mixtures comprising cancer cells,infected cells, and/or hematological cells.

In certain embodiments, a CD20-binding protein or pharmaceuticalcomposition of the present invention can be used to kill cancer cells ina mixture of different cell types, such as within an organism. Incertain embodiments, a CD20-binding protein or pharmaceuticalcomposition of the present invention, alone or in combination with othercompounds or pharmaceutical compositions can show potent cell-killactivity when administered to a population of cells, in vitro or in vivoin a subject such as in a patient in need of treatment. By targeting thedelivery of enzymatically active Shiga toxin regions using high-affinityimmunoglobulin-type binding regions to CD20, this potent cell-killactivity can be restricted to specifically and selectively kill certaincell types within an organism, such as cancer cells, neoplastic cells,malignant cells, non-malignant tumor cells, or infected cells.

The term “cancer cell” or “cancerous cell” refers to various neoplasticcells which grow and divide in an abnormally accelerated fashion andwill be clear to the skilled person. The term “cancer cell” includesboth malignant and non-malignant cells. Generally, cancers and/or tumorscan be defined as diseases, disorders, or conditions that are amenableto treatment and/or prevention. The cancers and tumors (either malignantor non-malignant) which are comprised by cancer cells and/or tumor cellswill be clear to the skilled person.

The present invention provides a method of killing a CD20 expressingcell in a patient, the method comprising the step of administering tothe patient at least one CD20-binding protein of the present inventionor a pharmaceutical composition thereof.

Certain embodiments of the CD20-binding protein or pharmaceuticalcompositions thereof can be used to kill a CD20 expressing immune cell(whether healthy or malignant) in a patient.

It is within the scope of the present invention to utilize theCD20-binding protein of the invention or pharmaceutical compositionthereof for the purposes of ex vivo depletion of B-cells from isolatedcell populations removed from a patient.

Additionally, the present invention provides a method of treating adisease, disorder, or condition in a patient comprising the step ofadministering to a patient in need thereof a therapeutically effectiveamount of at least one of the CD20-binding proteins of the presentinvention or a pharmaceutical composition thereof. Contemplateddiseases, disorders, and conditions that can be treated using thismethod include cancers, malignant tumors, non-malignant tumors, and,immune disorders. Administration of a “therapeutically effective dosage”of a compound of the invention can result in a decrease in severity ofdisease symptoms, an increase in frequency and duration of diseasesymptom-free periods, or a prevention of impairment or disability due tothe disease affliction.

The therapeutically effective amount of a compound of the presentinvention will depend on the route of administration, the type of mammalbeing treated, and the physical characteristics of the specific patientunder consideration. These factors and their relationship to determiningthis amount are well known to skilled practitioners in the medical arts.This amount and the method of administration can be tailored to achieveoptimal efficacy, and may depend on such factors as weight, diet,concurrent medication and other factors, well known to those skilled inthe medical arts. The dosage sizes and dosing regimen most appropriatefor human use may be guided by the results obtained by the presentinvention, and may be confirmed in properly designed clinical trials. Aneffective dosage and treatment protocol may be determined byconventional means, starting with a low dose in laboratory animals andthen increasing the dosage while monitoring the effects, andsystematically varying the dosage regimen as well. Numerous factors maybe taken into consideration by a clinician when determining an optimaldosage for a given subject. Such considerations are known to the skilledperson.

An acceptable route of administration may refer to any administrationpathway known in the art, including but not limited to aerosol, enteral,nasal, ophthalmic, oral, parenteral, rectal, vaginal, or transdermal(e.g. topical administration of a cream, gel or ointment, or by means ofa transdermal patch). “Parenteral administration” is typicallyassociated with injection at or in communication with the intended siteof action, including intratumoral injection, infraorbital, infusion,intraarterial, intracapsular, intracardiac, intradermal, intramuscular,intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal,intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous,transmucosal, or transtracheal administration.

For administration of a pharmaceutical composition of the invention, thedosage range will generally be from about 0.0001 to 100 milligrams perkilogram (mg/kg), and more usually 0.01 to 5 mg/kg, of the host bodyweight. Exemplary dosages may be 0.25 mg/kg body weight, 1 mg/kg bodyweight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg body weightor within the range of 1-10 mg/kg. An exemplary treatment regime is aonce or twice daily administration, or a once or twice weeklyadministration, once every two weeks, once every three weeks, once everyfour weeks, once a month, once every two or three months or once everythree to 6 months. Dosages may be selected and readjusted by the skilledhealth care professional as required to maximize therapeutic benefit fora particular patient.

Pharmaceutical compositions of the invention will typically beadministered to the same patient on multiple occasions. Intervalsbetween single dosages can be, for example, 2-5 days, weekly, monthly,every two or three months, every six months, or yearly. Intervalsbetween administrations can also be irregular, based on regulating bloodlevels or other markers in the subject or patient. Dosage regimens for acompound of the invention include intravenous administration of 1 mg/kgbody weight or 3 mg/kg body weight with the compound administered everytwo to four weeks for six dosages, then every three months at 3 mg/kgbody weight or 1 mg/kg body weight.

A pharmaceutical composition of the present invention may beadministered via one or more routes of administration, using one or moreof a variety of methods known in the art. As will be appreciated by theskilled worker, the route and/or mode of administration will varydepending upon the desired results. Routes of administration forCD20-binding proteins or pharmaceutical compositions of the inventioninclude, e.g. intravenous, intramuscular, intradermal, intraperitoneal,subcutaneous, spinal, or other parenteral routes of administration, forexample by injection or infusion at or in communication with theintended site of action (e.g. intratumoral injection). In otherembodiments, a CD20-binding protein or pharmaceutical composition of theinvention may be administered by a non-parenteral route, such as atopical, epidermal or mucosal route of administration, for example,intranasally, orally, vaginally, rectally, sublingually, or topically.

Therapeutic CD20-binding proteins or pharmaceutical compositions of theinvention may be administered with one or more of a variety of medicaldevices known in the art. For example, in one embodiment, apharmaceutical composition of the invention may be administered with aneedleless hypodermic injection device. Examples of well-known implantsand modules useful in the present invention are in the art, includinge.g., implantable micro-infusion pumps for controlled rate delivery;devices for administering through the skin; infusion pumps for deliveryat a precise infusion rate; variable flow implantable infusion devicesfor continuous drug delivery; and osmotic drug delivery systems. Theseand other such implants, delivery systems, and modules are known tothose skilled in the art.

A CD20-binding protein or pharmaceutical composition of the presentinvention may be administered alone or in combination with one or moreother therapeutic or diagnostic agents. A combination therapy mayinclude a CD20-binding protein of the invention or pharmaceuticalcomposition thereof combined with at least one other therapeutic agentselected based on the particular patient, disease or condition to betreated. Examples of other such agents include, inter alia, a cytotoxic,anti-cancer or chemotherapeutic agent, an anti-inflammatory oranti-proliferative agent, an antimicrobial or antiviral agent, growthfactors, cytokines, an analgesic, a therapeutically active smallmolecule or polypeptide, a single chain antibody, a classical antibodyor fragment thereof, or a nucleic acid molecule which modulates one ormore signaling pathways, and similar modulating therapeutics which mightcomplement or otherwise be beneficial in a therapeutic or prophylactictreatment regimen.

Treatment of a patient with certain embodiments of the CD20-bindingproteins or pharmaceutical compositions of the present invention willlead to cell death of targeted cells and/or the inhibition of growth oftargeted cells. As such, CD20-binding proteins of the invention, andpharmaceutical compositions comprising them, will be useful in methodsfor treating a variety of pathological disorders in which killing ordepleting target cells might be beneficial, such as, inter alia, cancer,immune disorders, and infected cells. The present invention providesmethods for suppressing cell proliferation, and treating cell disorders,including neoplasia and overactive B-cells.

In certain embodiments, CD20-binding proteins and pharmaceuticalcompositions of the invention can be used to treat or prevent cancers,tumors (malignant and non-malignant), and immune disorders.

In certain embodiments, the present invention provides methods fortreating malignancies or neoplasms and other blood cell-associatedcancers in a mammalian subject, such as a human, the method comprisingthe step of administering to a subject in need thereof a therapeuticallyeffective amount of a CD20-binding protein or pharmaceutical compositionof the invention.

The CD20-binding proteins and pharmaceutical compositions of theinvention have varied applications, including, e.g., uses asanti-neoplastic agents, uses in modulating immune responses, uses inpurging transplantation tissues of unwanted cell types, and uses asdiagnostic agents. The CD20-binding proteins and pharmaceuticalcompositions of the present invention are commonly anti-neoplasticagents—meaning they are capable of treating and/or preventing thedevelopment, maturation, or spread of neoplastic or malignant cells byinhibiting the growth and/or causing the death of cancer or tumor cells.

In certain embodiments, a CD20-binding protein or pharmaceuticalcomposition of the present invention is used to treat a B-cell-mediateddisease or disorder, such as for example leukemia, lymphoma, myeloma,amyloidosis, ankylosing spondylitis, asthma, Crohn's disease, diabetes,graft rejection, graft-host disease, Hashimoto's thyroiditis, hemolyticuremic syndrome, HIV-related diseases, lupus erythematosus, multiplesclerosis, polyarteritis, psoriasis, psoriatic arthritis, rheumatoidarthritis, scleroderma, septic shock, Sjörgren's syndrome, ulcerativecolitis, and vasculitis.

The CD20-binding proteins and pharmaceutical compositions of the presentinvention can be utilized in a method of treating cancer comprisingadministering to a patient, in need thereof, a therapeutically effectiveamount of the CD20-binding protein or a pharmaceutical composition ofthe present invention. Some cancers shown to have expression of CD20include, but are not limited to, B-cell lymphomas (including bothnon-Hodgkin's and Hodgkin's), hairy cell leukemia, B-cell chroniclymphocytic leukemia, some T-cell lymphomas, and melanoma cancer stemcells. In certain embodiments of the methods of the present invention,the cancer being treated is selected from the group consisting of bonecancer, leukemia, lymphoma, melanoma, and myeloma.

The CD20-binding proteins and pharmaceutical compositions of the presentinvention can be utilized in a method of treating an immune disordercomprising administering to a patient, in need thereof, atherapeutically effective amount of the CD20-binding protein or apharmaceutical composition of the present invention. In certainembodiments of the methods of the present invention, the immune disorderis related to an inflammation associated with a disease selected fromthe group consisting of: amyloidosis, ankylosing spondylitis, asthma,Crohn's disease, diabetes, graft rejection, graft-versus host disease,Hashimoto's thyroiditis, hemolytic uremic syndrome, HIV-relateddiseases, lupus erythematosus, multiple sclerosis, polyarteritis,psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma,septic shock, Sjörgren's syndrome, ulcerative colitis, and vasculitis.

Among certain embodiments of the present invention is using theCD20-binding protein as a component of a medicament for the treatment orprevention of a cancer, tumor, or immune disorder. For example, immunedisorders presenting on the skin of a patient may be treated with such amedicament in efforts to reduce inflammation.

Beyond the CD20-binding proteins of the present invention, thepolynucleotides which encode such molecules, when applicable, are withinthe scope of the present invention. The term “polynucleotides” isequivalent to the term “nucleic acids” both of which include polymers ofdeoxyribonucleic acids and ribonucleic acids. Such polynucleotides arespecifically disclosed to include all polynucleotides capable ofencoding a specified CD20-binding protein, for example, taking intoaccount the wobble known to be tolerated in the third position of aminoacid codons, yet encoding for an equivalent amino acid. Further, thepresent invention comprises expression vectors that comprise thepolynucleotides within the scope of the invention. Such expressionvectors will include the polynucleotides necessary to support productionof the CD20-binding proteins of the invention within any host cell ofchoice. The specific polynucleotides comprising expression vectors foruse with specific types of host cells are well known to one of ordinaryskill in the art, can be determined using routine experimentation, ormay be purchased.

The present invention also provides methods of rapidly internalizing theCD20-binding protein into the interior of a cell, by contacting the cellwith a CD20-binding protein of the invention either in vivo or in vitro,such as within a patient. The present invention further provides methodsof killing a CD20 expressing cell, where that cell expresses a CD20antigen on its surface, by contacting the cell with a CD20-bindingprotein of the invention either in vivo or in vitro, such as within apatient.

If the CD20-binding proteins of the present invention comprise or areconjugated to exogenous material, as described above, those CD20-bindingproteins can be utilized in a method of delivering that exogenousmaterial into a target cell that expresses a CD20 antigen on its cellsurface. The present invention also provides methods of deliveringexogenous materials into the interior of a CD20 expressing cell, bycontacting the cell with a CD20-binding protein of the invention eitherin vivo or in vitro, such as within a patient.

Additionally, the CD20-binding proteins of the invention can be utilizedin a method for treating cancer, wherein the tumor or cancer cellexpresses on its surface a CD20 antigen, which method comprisesadministering the protein of the present invention to a patient in needof such treatment. Some cancers shown to have expression of CD20include, but are not limited to, B-cell lymphomas (including bothnon-Hodgkin's and Hodgkin's), hairy cell leukemia, B-cell chroniclymphocytic leukemia, some T-cell lymphomas, and melanoma cancer stemcells.

For purposes of the present invention, the term “lymphoma” includesB-cell lymphomas (such as non-Hodgkin's and Hodgkin's types), hairy cellleukemia, B-cell chronic lymphocytic leukemia, T-cell lymphomas, andmelanoma cancer stem cell type lymphomas.

Certain embodiments of the invention are below, numbered 1-40 andreferring to Table C for biological sequences: (1) A CD20-bindingprotein for the internalization of the CD20 antigen in a cell, whereinthe protein comprises a binding region specific for CD20 and a toxineffector region derived from Shiga-like toxin 1 (SLT-1), wherein theprotein induces rapid internalization of CD20 present on the surface ofthe cell. (2) The CD20-binding protein of embodiment 1, wherein theprotein induces internalization of CD20 in a B-cell lineage cell in lessthan about one hour. (3) The CD20-binding protein of claim 1, whereinthe toxin effector region comprises amino acids 75 to 251 of NO:1 (seeTable C). (4) The CD20-binding protein of embodiment 1, wherein thetoxin effector region comprises amino acids 1 to 251 of NO:1. (5) TheCD20-binding protein of embodiment 1, wherein the toxin effector regioncomprises amino acids 1 to 261 of NO:1. (6) The CD20-binding protein ofembodiment 1, wherein the protein is cytotoxic.

(7) The CD20-binding protein of embodiment 1, wherein the CD20 bindingregion is selected from the group consisting of an Fab fragment, anF(ab′)2 fragment, an Fd fragment, an Fv fragment a dAb fragment, a scFv,a diabody, a CDR3 peptide, a constrained FR3-CDR3-FR4 peptide, ananobody, a bivalent nanobody, small modular immunopharmaceuticals(SMIPs), a shark variable IgNAR domain, a minibody, and any fragment orchemically or genetically manipulated counterparts that retain CD20binding function. (8) The CD20-binding protein of embodiment 1, whereinthe binding region is a scFv.

(9) The CD20-binding protein of embodiment 8, wherein the binding regioncomprises (A) (i) a heavy chain variable (VH) domain comprising HCDR1,HCDR2, HCDR3 amino acid sequences as shown in NO:6, NO:7, and NO:8,respectively, and (ii) a light chain variable (VL) domain comprisingLCDR1, LCDR2, and LCDR3 amino acid sequences as shown in NO:9, NO:10,and NO:11, respectively; or (B) (i) a heavy chain variable (VH) domaincomprising HCDR1, HCDR2, and HCDR3 amino acid sequences as shown inNO:21, NO:22, and NO:23, respectively, and (ii) a light chain variable(VL) domain comprising LCDR1, LCDR2, and LCDR3 amino acid sequences asshown in NO:24, NO:10, and NO:11, respectively.

(10) The CD20-binding protein of embodiment 8, wherein the CD20 bindingregion comprises amino acids 2 to 245 of NO:4. (11) The CD20-bindingprotein of embodiment 1, wherein the CD20 binding region comprises aminoacids 2 to 245 of NO: 4 and the toxin effector region comprises aminoacids 75 to 251 of NO:1. (12) The CD20-binding protein of embodiment 1,which comprises NO:4. (13) A CD20-binding protein for killing a cellwhich expresses CD20 on its surface wherein the binding region comprisesa heavy chain variable (VH) domain comprising HCDR1, HCDR2, and HCDR3amino acid sequences as shown in NO:6, NO:7, and NO:8, respectively, anda light chain variable (VL) domain comprising LCDR1, LCDR2, and LCDR3amino acid sequences as shown in NO:9, NO:10, and NO:11, respectively,whereby upon administration, the protein is capable of killing a cellwhich expresses CD20 on its surface.

(14) The CD20 binding-protein of embodiment 13, wherein the CD20 bindingregion comprises amino acids 2 to 245 of NO:4. (15) The CD20binding-protein of embodiment 13, wherein the CD20 binding regioncomprises amino acids 2 to 245 of NO: 4 and the toxin effector regioncomprises amino acids 75 to 251 of NO:1. (16) The CD20 binding-proteinof embodiment 13 which comprises NO:4.

(17) A CD20 binding-protein for the delivery of exogenous material intothe cell that expresses CD20 on its surface, wherein the proteincomprises a binding region specific for CD20, a toxin effector regionwherein said toxin effector region is derived from Shiga-like toxin 1(SLT-1), and the exogenous material, whereby upon administration, theprotein is capable of delivering the exogenous material into a cellwhich expresses CD20 on its surface.

(18) The CD20-binding protein of embodiment 17, wherein the bindingregion comprises (A) (i) a heavy chain variable (VH) domain comprisingHCDR1, HCDR2, HCDR3 amino acid sequences as shown in NO:6, NO:7, andNO:8, respectively, and (ii) a light chain variable (VL) domaincomprising LCDR1, LCDR2, and LCDR3 amino acid sequences as shown inNO:9, NO:10, and NO:11, respectively; or (B) (i) a heavy chain variable(VH) domain comprising HCDR1, HCDR2, and HCDR3 amino acid sequences asshown in NO:21, NO:22, and NO:23, respectively, and (ii) a light chainvariable (VL) domain comprising LCDR1, LCDR2, and LCDR3 amino acidsequences as shown in NO:23, NO:10, and NO:11, respectively.

(19) The CD20-binding protein of embodiment 18, wherein the exogenousmaterial is selected from the group consisting of a peptide, a protein,and a nucleic acid. (20) The CD20 binding-protein of embodiment 19,wherein the exogenous material is a peptide and the peptide is anantigen. (21) The CD20 binding-protein of embodiment 20, wherein theantigen is encoded between the binding region and the toxin effectorregion of the protein. (22) The CD20 binding-protein of embodiment 19wherein the antigen is derived from a viral protein. (23) The CD20binding-protein of embodiment 21 wherein the antigen is NO:2. (24) TheCD20 binding-protein of embodiment 21 comprising NO:5. (25) The CD20binding-protein of embodiment 20, wherein the antigen is derived from abacterial protein. (26) The CD20 binding-protein of embodiment 20,wherein the antigen is derived from a protein mutated in cancer. (27)The CD20 binding-protein of embodiment 20, wherein the antigen isderived from a protein aberrantly expressed in cancer. (28) The CD20binding-protein of embodiment 20, wherein the antigen is derived from aT-cell CDR region.

(29) The CD20 binding-protein of embodiment 19, wherein the exogenousmaterial is a protein. (30) The CD20 binding-protein of embodiment 29,wherein the protein is an enzyme. (31) The CD20 binding-protein ofembodiment 19 wherein the exogenous material is a nucleic acid. (31) TheCD20 binding-protein of embodiment 30 wherein the nucleic acid is asiRNA. (32) A polynucleotide that encodes the CD20 binding-protein ofembodiment 1. (33) An expression vector that comprises thepolynucleotide of embodiment 32. (34) A host cell comprising theexpression vector of embodiment 33.

(35) A method of rapidly internalizing the CD20 antigen into the cell ofa patient, the method comprising the step of administering to thepatient a protein of any one of embodiments 1-12. (36) A method ofkilling a cell in a patient expressing the CD20 antigen on its surface,the method comprising the step of administering to a patient a proteinof any of embodiments 1-16. (37) A method of delivering exogenousmaterial into a cell of a patient that expresses CD20 on its surface,the method comprising the step of administering to the patient a proteinof any one of embodiments 17-31. (39) A method of treating cancer in apatient, wherein the cancer expresses on the tumor or cancer cellsurface a CD20 antigen, the method comprising the step of administeringto the patient a protein of any one of embodiments 1-31. (40) The methodof embodiment 39 wherein the cancer is lymphoma.

TABLE C Sequences referred to in embodiments 1-40 Text NumberDescription Sequence NO: 1 SLT-1 A KEFTLDFSTAKTYVDSLNVIRSA subunitIGTPLQTISSGGTSLLMIDSGSG polypeptide DNLFAVDVRGIDPEEGFNNLRLIVERNNLYVTGFVNRTNNVFYRFA SHVTFPGTTAVTLSGDSSYTTLQ RVAGISRTGMQINRHSLTTSYLDLMSFISGTSLTQSVARAMLRFVT VTAEALRFRQIQRGFRTTLDDLS GRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAT LGSVALILNCHHHARVARMASDE FPSMCPADGRVRGITHNKILWDSSTLGAILMRRTISS NO: 2 SLT-1 A aargarttyacnytngayttyws subunitnacngcnaaracntaygtngayw polynucleotide snytnaaygtnathmgnwsngcn(consensus) athggnacnccnytncaracnat hwsnwsnggnggnacnwsnytnytnatgathgaywsnggnwsnggn gayaayytnttygcngtngaygt nmgnggnathgayccngargarggnmgnttyaayaayytnmgnytn athgtngarmgnaayaayytnta ygtnacnggnttygtnaaymgnacnaayaaygtnttytaymgntty gcngayttywsncaygtnacntt yccnggnacnacngcngtnacnytnwsnggngaywsnwsntayacn acnytncarmgngtngcnggnat hwsnmgnacnggnatgcarathaaymgncaywsnytnacnacnwsn tayytngayytnatgwsncayws nggnacnwsnytnacncarwsngtngcnmgngcnatgytnmgntty gtnacngtnacngcngargcnyt nmgnttymgncarathcarmgnggnttymgnacnacnytngaygay ytnwsnggnmgnwntaygtnatg acngcngargaygtngayytnacnytnaaytggggnmgnytnwsnw sngtnytnccngaytaycayggn cargaywsngtnmgngtnggnmgnathwsnttyggnwsnathaayg cnathytnggnwsngtngnytna thytnaaytgycaycaycaygcnwsnmgngtngcnmgnatggcnws ngaygarttyccnwsnatgtgyc cngcngayggnmgngtnmgnggnathacncayaayaarathytntg ggaywsnwsnacnytnggngcna thytnatgmgnmgnacnathwsnwsn NO: 3 Influenza GILGFVFTL Matrix 58-66 NO: 4 MT-3724QVQLQQPGAELVKPGASVKMSCK polypeptide TSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKAT LTADKSSSTVYMQLSSLTSEDSA VYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGS QIVLSQSPTILSASPGEKVTMTC RASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTS YSLTISRVEAEDAATYYCQQWIS NPPTFGAGTKLELKEFPKPSTPPGSSGGAPKEFTLDFSTAKTYVDS LNVIRSAIGTPLQTISSGGTSLL MIDSGSGDNLFAVDFRGIDPEEGRFNNLRLIVERNNLYVTGFVNRT NNVFYRFADFSHVTFPGTTAVTL SGDSSYTTLQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQSV ARAMLRFVTVTAEALRFRQIQRG FRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGR ISFGSINAILGSVALILNCHHHA SRVAR NO: 5 MT-3724cargtncarytncarcarccngg polynucleotide ngcngarytngtnaarccnggng consensuscnwsngtnaaratgwsntgyaar acnwsnggntayacnttyacnws ntayaaygtncaytgggtnaarcaracnccnggncarggnytngar tggathggngcnathtayccngg naayggngayacnwsnttyaaycaraarttyaarggnaargcnacn ytnacngcngayaarwsnwsnws nacngtntayatgcarytnwsnwsnytnacnwsngargaywsngcn gtntaytgygcnmgnwsnaayta ytayggnwsnwsntaygtntggttyttygaygtntggggngcnggn acnacngtnacngtnwsnwsngg nwsnacnwsnggnwsnggnaarccnggnwsnggngarggnwsncar athgtnytnwsncarwsnccnac nathytnwsngcnwsnccnggngaraargtnacnatgacntgymgn gcnwsnwsnwsngtnwsntayat ggaytggtaycarcaraarccnggnwsnwsnccnaarccntggath taygcnacnwsnaayytngcnws nggngtnccngcnmgnttywsnggnwsnggnwsnggnacnwsntay wsnytnacnathwsnmgngtnga rgcngargaygcngcnacntaytaytgycarcartggathwsnaay ccnccnacnttyggngcnggnac naarytngarytnaargarttyccnaarccnwsnacnccnccnggn wsnwsnggnggngcnccnaarga rttyacnytngayttywsnacngcnaaracntaygtngaywsnytn aaygtnathmgnwsngcnathgg nacnccnytncaracnathwsnwsnggnggnacnwsnytnytnatg athgaywsnggnwsnggngayaa yytnttygcngtngaygtnmgnggnathgayccngargarggnmgn ttyaayaayytnmgnytnathgt ngarmgnaayaayytntaygtnacnggnttygtnaaymgnacnaay aaygtnttytaymgnttygcnga yttywsncaygtnacnttyccnggnacnacngcngtnacnytnwsn ggngaywsnwsntayacnacnyt ncarmgngtngcnggnathwsnmgnacnggnatgcarathaaymgn caywsnytnacnacnwsntayyt ngayytnatgwsncaywsnggnacnwsnytnacncarwsngtngcn mgngcnatgytnmgnttygtnac ngtnacngcngargcnytnmgnttymgncarathacrmgnggntty mgnacnytngayfayytnwsngg nmgnwsntaygtnatgacngcngargaygtngayytnacnytnaay tggggnmgnytnwsnwsngtnyt nccngaytaycayggncargaywsngtnmgngtnggnmgnathwsn ttyggnwsnathaaygcnathyt nggnwsngtngcnytnathytnaaytgycaycaycaygcnwsnmgn gtngcnmgn NO: 6 Heavy chain GYTFTSYNVH CDR1NO: 7 Heavy chain AIYPGNGDTSFNQKFKG CDR2 NO: 8 Heavy chainSNYYGSSYVWFFDY CDR3 NO: 9 Light chain RASSSVSYMD CDR1 NO: 10 Light chainATSNLAS CDR2 NO: 11 Light chain QQWISNPPT CDR3 NO: 12 B9E9-SLTAQVQLVQSGAELVKPGASVKMSCK polypeptide ASGYTETSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKAT LTADKSSSTAYMQLSSLTSEDSA VYYCARAQLRPNYWYFDVWGAGTTVTVSSGGGGSGGGGSGGGGSGG GGSGGGGSDIVLSQSPAILSASP GEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLASGVPARF SGSGSGTSYSLTISRVEAEDAAT YYCQQWISNPPTEGAGTKLELKGGGGSGGKEFTLDESTAKTYVDSL NVIRSAIGTPLQTISSGGTSLLM IDSGSGDNLEAVDVRGIDPEEGRENNLRLIVERNNLYVTGEVNRIN NVEYREADESHVTFPGITAVTLS GDSSYTTLQRVAGISRTGMQINRHSLITSYLDLNISHSGTSLTQSV ARAMERFVTVTAEAERFRQIQRG ERTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGR ISEGSINAILGSVALILNCHMIA SRVAR NO: 13 B9E9-SLTAcargtncarytngtncarwsngg polynucleotide ngcngarytngtnaarccnggng consensuscnwsngtnaaratgwsntgyaar gcnwsnggntayacnttyacnws ntayaayatgcaytgggtnaarcaracnccnggncarggnytngar tggathggngcnathtayccngg naayggngayacnwsntayaaycaraarttyaarggnaargcnacn ytnacngcngayaarwsnwsnws nacngcntayatgcarytnwsnwsnytnacnwsngargaywsngcn gtntaytaytgygcnmgngcnca rytnmgnccnaaytaytggtayttygaygtntggggngcnggnacn acngtnacngtnwsnwsnggngg nggnggnwsnggnggnggnggnwsnggnggnggnggnwsnggnggn ggnggnwsnggnggnggnggnws ngayathgtnytnwsncarwsnccngcnathytnwsngcnwsnccn ggngaraargtnacnatgacntg ymgngcnwsnwsnwsngtnwsntayatgcaytggtaycarcaraar ccnggnwsnwsnccnaarccntg gathtaygcnacnwsnaayytngcnwsnggngtnccngcnmgntty wsnggnwsnggnwsnggnacnws ntaywsnytnacnathwsnmgngtngargcngargaygcngcnacn taytaytgycarcartggathws naayccnccnacnttyggngcnggnacnaarytngarytnaarggn ggnggnggnwsnggnggnaarga rttyacnytngayttywsnacngcnaaracntaygtngaywsnytn aaygtnathmgnwsngcnathgg nacnccnytncaracnathwsnwsnggnggnacwsnytnytnatga thgaywsnggnwsnggngayaay ytnttygcngtngaygtnmgnggnathgayccngargarggnmgnt tyaayaayytnmgnytnathgtn garmgnaayaayytntaygtnacnggnttygtnaaymgnacnaaya aygtnttytaymgnttygcngay ttywsncaygtnacnttyccnggnacnacngcngtnacnytnwsng gngaywsnwsntayacnacnytn carmgngtngcnggnathwsnmgnacnggnatgcarathaaymgnc aywsnytnacnacnwsntayytn gayytnatgwsncaywsnggnacnwsnytnacncarwsngtngcnm gngcnatgytnmgnttygtnacn gcngargcnytnmgnttymgncarathcarmgnggnttymgnacna cnytngaygayytnwsnggnmgn wsntaygtnatgacngcngargaygtngayytnacnytnaaytggg gnmgnytnwsnwsngtnytnccn gaytaycayggncargaywsngtnmgngtnggnmgnathwsnttyg gnwsnathaaygcnathytnggn wsngtngcnytnathytnaaytgycaycaycaygcnwsnmgngtng cnmgn NO: 14 C2B8-SLTA QVQLQQPGAELVKPGASVKMSCKpolypeptide ASGYTFTSYNMHWVKQTPGRGLE WIGAIYPGNGDTSYNQKFKGKATLTADIKSSSTAYMQLSSLTSEDS AVYYCARSTYYGGDWYFNVWGAG TTVTVSAGSTSGSGKPGSGEGSTKGQIVLSQSPAILSASPGEKVTM TCRASSSVSYIHWFQQKPGSSPK PWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQW TSNPPTFGGGTKLEIKEFPKPST PPGSSGGAPKEETLDFSTAKTYVDSLNVIRSAIGTPLQTESSGGTS LLMIDSGSGDNLFAVDVRGIDPE EGRFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAV TLSGDSSYTTLQRVAGISRTGMQ INRHSLTTSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQ RGFRTTLDDLSGRSYVMTAEDVD LTENWGRESSVLPDYIIGQDSVRVGRISFGSINAILGSVALILNCH HHASRVAR NO: 15 C2B8-SLTAcargtncarytncarcarccngg polynucleotide ngcngarytngtnaarccnggng consensuscnwsngtnaaratgwsntgyaar gcnwsnggntayacnttyacnws ntayaayatgcaytgggtnaarcaracnccnggnmgnggnytngar tggathggngcnathtayccngg naayggngayacnwsntayaaycaraarttyaarggnaargcnacn ytnacngcngayaarwsnwsnws nacngcntayatgcarytnwsnwsnytnacnwsngargaywsngcn gtntaytaytgygcnmgnwsnna cntaytayggnggngaytggtayttyaaygtntggggngcnggnac nacngtnacngtnwsngcnggnw snacnwsnggnwsnggnaarccnggnwsnggngarggnwsnacnaa rggncarathgtnytnwsncarw snccngcnathytnwsngcnwsnccnggngaraargtnacnatgac ntgymgngcnwsnwsnwsngtnw sntayathcaytggttycarcaraarccnggnwsnwsnccnaarcc ntggathtaygcnacnwsnaayy tngcnwsnggngtnccngtnmgnttywsnggnwsnggnwsnggnac nwsntaywsnytnacnathwsnm gngtngargcngargaygcngcnacntaytaytgycarcartggac nwsnaayccnccnacnttyggng gnggnacnaarytngarathaargarttyccnaarccnwsnacncc nccnggnwsnwsnggnggngcnc cnaargarttyacnytngayttywsnacngcnaaracntaygtnga ywsnytnaaygtnathmgnwsng cnathggnacnccnytncaracnathwsnwsnggnggnacnwsnyt nytnatgathgaywsnggnwsng gngayaayytnttygcngtngaygtnmgnggnathgayccngarga rggnmgnttyaayaayytnmgny tnathgtngarmgnaayaayytntaygtnacnggnttygtnaaymg nacnaayaaygtnttytaymgnt tygcngayttywsncaygtnacnttyccnggnacnacngcngtnac nytnwsnggngaywsnwsntaya cnacnytncarmgngtngcnggnathwsnmgnacnggnatncarat haaymgncaywsnytnacnacnw sntayytngayytnatgwsncaywsnggnacnwsnytnacncarws ngtngcnmgngcnatgytnmgnt tygtnacngtnacngcngargcnytnmgnttymgncarathcarmg nggnttymgnacnacnytngayg ayytnwsnggnmgnwsntaygtnatgacngcngargaygtngayyt nacnytnaaytggggnmgnytnw snwsngtnytnccngaytaycayggncargaywsngtnmgngtngg nmgnathwsnttyggnwsnatha aygcnathytnggnwsngtngcnytnathytnaaytgycaycayca ygcnwsnmgngtngcnmgn NO: 16 MT-3727QVQLQQPGAELVKPGASVKMSCK polypeptide TSGYTFTSYNVHWVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKAT LTADKSSSTVYMQLSSLTSEDSA VYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGSTSGSGKPGSGEGS QIVLSQSPTILSASPGEKVTMTC RASSSVSYMDWYQQKPGSSPKPWIYATSNLASGVPARFSGSGSGTS YSLTISRVEAEDAATYYCQQWIS NPPTFGAGTKLELKEEPKPSTPPGSSGGAPGILGFVFTLKEFTLDE STAKTYVDSENVIRSAIGTPLQT ISSGGTSLLMIDSGSGDNLEAVDVRGIDPEEGRENNLRLIVIERNN LYVTGIFVNRTNNVEYREADFSI TVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRITSLTTSYLD LMSHSGTSLTQSVARAMLRFVTV TAEALRERQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRLSSVL PDYHGQDSVRVGRISFGSINAIL GSVALIENCHHHASRVARNO: 17 MT-3727 cargtncarytncarcarccngg polynucleotidengcngarytngtnaarccnggng consensus cnwsngtnaaratgwsntgyaaracnwsnggntayacnttyacnws ntayaaygtncaytgggtnaarc aracnccnggncarggnytngartggathggngcnathtayccngg naayggngayacnwsnttyaayc araarttyaarggnaargcnacnytnacngcngayaarwsnwsnws nacngtntayatgcarytnwsnw snytnacnwsngargaywsngcngtntaytaytgygcnmgnwsnaa ytaytayggnwsnwsntaygtnt ggttyttygaygtntggggngcnggnacnacngtnacngtnwsnws nggnwsnacnwsnggnwsnggna arccnggnwsnggngarggnwsncarathgtnytnwsncarwsncc nacathytnwsngcnwsnccngg ngaraargtnacnatgacntgymgngcnwsnwsnwsngtnwsntay atggaytggtaycarcaraarcc nggnwsnwsnccnaarccntggathtaygcnacnwsnaayytngcn wsnggngtnccngcnmgnttyws nggnwsnggnwsnggnacnwsntaywsnytnacnathwsnmgngtn gargcngargaygcngcnacnta ytaytgycarcartggathwsnaayccnccnacnttyggngcnggn acnaarytngarytnaargartt yccnaarccnwsnacnccnccnggnwsnwsnggnggngcnccnggn athytnggnttygtnttyacnyt naargarttyacnytngayttywsnacngcnaaracntaygtngay wsnytnaaygtnathmgnwsngc nathggnacnccnytncaracnathwsnwsnggnggnacnwsnytn ytnatgathgaywsnggnwsngg ngayaayytnttygcngtngaygtnmgnggnathgayccngargar ggnmgnttyaayaayytnmgnyt nathgtngarmgnaayaayytntaygtnacnggnttygtnaaymgn acnaayaaygtnttytaymgntt ygcngayttywsncaygtnacnttyccnggnacnacngcngtnacn ytnwsnggngaywsnwsntayac nacnytncarmgngtngcnggnathwsnmgnacnggnatgcarath aaymgncaywsnytnacnacnws ntayytngayytnatgwsncaywsnggnacnwsnytnacncarwsn gtngcnmgngcnatgytnmgntt ygtnacngtnacngcngargcnytnmgnttymgncarathcarmgn ggnttymgnacnacnytngayga yytnwsnggnmgnwsntaygtnatgacngcngargaygtngayytn acnytnaaytggggnmgnytnws nwsngtnytnccngaytaycayggncargaywsngtnmgngtnggn mgnathwsnttyggnwsnathaa ygcnathytnggnwsngtngcnytnathytnaaytgycaycaycay gcnwsnmgngtngcnmgn NO: 18 218 LinkerGSTSGSGKPGSGEGS NO: 19 Strep leader MWSHPQFEK sequence NO: 20Murine IgG3 EFPKPSTPPGSSGGAP (mhinge) NO: 21 Heavy chain GYTFTSYNMH CDR1NO: 22 Heavy chain AIYPGNGDTSYNQKFKG CDR2 NO: 23 Heavy chainAQLRPNYWYFDV CDR3 NO: 24 Light chain RASSSVSYMH CDR1

The present invention is further illustrated by the followingnon-limiting examples of CD20-binding proteins comprising Shiga toxineffector regions derived from A Subunits of members of the Shiga toxinfamily and CD20 binding regions comprising immunoglobulin-typepolypeptides capable of binding extracellular parts of CD20.

EXAMPLES

The following examples demonstrate certain embodiments of the presentinvention. However, it is to be understood that these examples are forillustration purposes only and do not intend, nor should any beconstrued, to be wholly definitive as to conditions and scope of thisinvention. The examples were carried out using standard techniques,which are well known and routine to those of skill in the art, exceptwhere otherwise described in detail.

The following examples demonstrate the ability of exemplary CD20-bindingproteins to selectively kill cells which express CD20 on their cellsurfaces. The exemplary CD20-binding proteins bound to extracellularantigens on CD20 expressed by targeted cell types and entered thetargeted cells. The internalized CD20-binding proteins routed theirShiga toxin effector region to the cytosol to inactivate ribosomes andsubsequently caused the apoptotic death of the targeted cells. Thus, theexemplary CD20-binding proteins were capable of internalizing withinCD20 expressing cell types by virtue of their Shiga toxin effectorregions inducing rapid cellular internalization after the CD20-bindingproteins formed a complex with cell surface CD20.

These exemplary CD20-binding proteins include αCD20scFv::SLT-1A version1 (SEQ ID NO:4), αCD20scFv::SLT-1A version 2 (SEQ ID NO:16), B9E9-SLT-1A(SEQ ID NO:12), and C2B8-SLT-1A (SEQ ID NO:14).

Example 1—Construction, Production, and Purification of ExemplaryCD20-Binding Proteins

First, a CD20 binding region and a Shiga toxin effector region weredesigned or selected. In the examples below, the Shiga toxin effectorregion was derived from the A subunit of Shiga-like Toxin 1 (SLT-1A). Apolynucleotide was obtained containing a fragment of SLT-1A cloned intothe pECHE9A plasmid and encoding amino acids 1-251 of SLT-1A (Cheung Met al., Mol Cancer 9: 28 (2010)).

The CD20 binding region was designed as a recombinant scFv derived fromthe 1H4 CD20 monoclonal antibody (Haisma et al. (1999), Blood 92:184-90). The two immunoglobulin variable regions (V_(L) and V_(H)) wereseparated by a linker (SEQ ID NO:18).

Second, the binding region and Shiga toxin effector region were combinedto form a single-chain, recombinant polypeptide. In this example, apolynucleotide encoding the recombinant scFv derived from 1H4 CD20monoclonal antibody was cloned in frame with a “murine hinge”polynucleotide derived from polynucleotides encoding a murine IgG3molecule (SEQ ID NO:20) and in frame with a polynucleotide encodingSLT-1A (residues 1-251 of SEQ ID NO:1). The full-length sequence beginswith Strep-Tag® (SEQ ID NO:19) encoding polynucleotide sequence clonedin frame to facilitate detection and purification. The polynucleotidesequence of this example was codon optimized for efficient expression inE. coli using services from DNA 2.0, Inc. (Menlo Park, Calif., U.S.) toproduce the expression vector which encoded αCD20scFv::SLT-1A version 1.

A different CD20-binding protein comprising an influenza antigen wasconstructed and produced in a similar manner. DNA 2.0, Inc. (Menlo Park,Calif., U.S.) synthesized the multiple polynucleotides, including theantigen sequence (SEQ ID NO:3) and the required polynucleotidecomponents were joined in frame using vector pJ201 to create the openreading frame coding for the following single-chain polypeptide (fromamino-terminus to carboxy-terminus) Strep-Tag® (SEQ ID NO:19), the1H4-derived recombinant scFv (described above), the murine IgG3 molecule(SEQ ID NO:20), the linker (SEQ ID NO:3), and the SLT-1A-derivedsequence (residues 1-251 of SEQ ID NO:1). This recombinantpolynucleotide was cloned into pTXB1 for polypeptide productionpurposes. Again, codon optimization for efficient expression in E. coliwas performed by DNA 2.0, Inc. (Menlo Park, Calif., U.S.) to produce theexpression vector which encoded αCD20scFv::SLT-1A version 2.

Third, both versions 1 and 2 of the αCD20scFv::SLT-1A recombinantCD20-binding proteins were produced by using standard techniques forboth bacterial and cell-free, protein translation systems. Then theCD20-binding proteins were purified and isolated using techniques wellknown in the art.

Example 2—Determining the Dissociation Constant (K_(D)) of ExemplaryCD20-Binding Proteins

The cell binding characteristics of both versions 1 and 2 of theαCD20scFv::SLT-1A CD20-binding proteins were determined by afluorescence-based flow cytometry assay. Each sample contained 0.5×10⁶of either CD20 expressing cells (Raji (CD20+)) or non-expressing cells(BC1 (CD20-)) and was incubated with 1004 of various dilutions of theCD20-binding proteins in phosphate buffered saline Hyclone 1×PBS (FisherScientific, Waltham, Mass.) with 1% bovine serum albumin (BSA)(Calbiochem, San Diego, Calif., U.S.), hereinafter referred to as“1×PBS+1% BSA” for 1 hour at 4 degrees Celsius (° C.). The highestconcentration of CD20-binding protein was selected to lead to saturationof the reaction. The cells were washed twice with 1×PBS+1% BSA. Thecells were incubated for 1 hour at 4° C. with 100 μL of 1×PBS+1% BSAcontaining 0.3 μg of anti-Strep Tag® mAb-FITC (# A01736-100, Genscript,Piscataway, N.J., U.S.). The cells were washed twice with 1×PBS+1% BSA,suspended in 2004 of 1×PBS, and subjected to flow cytometry. Thebaseline corrected mean fluorescence intensity (MFI) data for all thesamples was obtained by subtracting the MFI of the FITC alone sample(negative control) from each experimental sample. Graphs were plotted ofMFI versus “concentration of protein” using Prism software (GraphPadSoftware, San Diego, Calif., U.S.). Using the Prism software function ofone-site binding [Y=B_(max)*X/(K_(D)+X)] under the headingbinding-saturation, the B_(max) and K_(D) were calculated using baselinecorrected data. B_(max) is the maximum specific binding reported in MFI.K_(D) is the equilibrium binding constant, reported in nanomolar (nM).

Over multiple experiments, the K_(D) of αCD20scFv::SLT-1A version 1 forRaji (CD20+) cells was determined to be about 80-100 nM. In oneexperiment, the B_(max) for the αCD20scFv::SLT-1A version 1 CD20-bindingprotein binding to CD20+ cells was measured to be about 140,000 MFI witha K_(D) of about 83 nM (Table 1), whereas there was no meaningfulbinding to CD20-cells observed in this assay. In one experiment, theB_(max) for αCD20scFv::SLT-1A version 2 binding to CD20+ cells wasmeasured to be about 110,000 MFI with a K_(D) of about 101 nM (Table 1),whereas there was no meaningful binding to CD20-cells observed in thisassay.

TABLE 1 Binding Characteristics: Representative values for B_(max) andK_(D) for exemplary CD20-binding proteins Target Target PositiveNegative Cells Cells target B_(max) K_(D) B_(max) K_(D) CD20-BindingProtein biomolecule (MFI) (nM) (MFI) (nM) αCD20scFv::SLT-1A CD20 139,00082.5 15,800 1,050 version 1 αCD20scFv::SLT-1A CD20 112,000 101.0 8,300280 version 2

Example 3—Determining the Half Maximal Inhibitory Concentration (IC₅₀)of Exemplary CD20-Binding Proteins

The ribosome inactivation capabilities of both versions 1 and 2 of theαCD20scFv::SLT-1A CD20-binding proteins were determined using acell-free, in vitro protein translation assay using the TNT® QuickCoupled Transcription/Translation kit (L1170 Promega Madison, Wis.,U.S.). The kit includes Luciferase T7 Control DNA (L4821 PromegaMadison, Wis., U.S.) and TNT® Quick Master Mix. The ribosome activityreaction was prepared according to the manufacturer's instructions.

A series of 10-fold dilutions of the αCD20scFv::SLT-1A version to betested was prepared in appropriate buffer and a series of identical TNTreaction mixture components were created for each dilution. Each samplein the dilution series of the αCD20scFv::SLT-1A proteins was combinedwith each of the TNT reaction mixtures along with the Luciferase T7Control DNA. The test samples were incubated for 1.5 hours at 30° C.After the incubation, Luciferase Assay Reagent (E1483 Promega, Madison,Wis., U.S.) was added to all test samples and the amount of luciferaseprotein translation was measured by luminescence according to themanufacturer instructions. The level of translational inhibition wasdetermined by non-linear regression analysis of log-transformedconcentrations of total protein versus relative luminescence units.Using statistical software (GraphPad Prism, San Diego, Calif., U.S.),the half maximal inhibitory concentration (IC₅₀) value was calculatedfor each sample using the Prism software function of log(inhibitor) vs.response (three parameters) [Y=Bottom+((Top−Bottom)/(1+10{circumflexover ( )}(X−Log IC50)))] under the heading dose-response-inhibition. TheIC₅₀ for experimental proteins and SLT-1A-only control protein werecalculated. The percent of SLT-1A-only control protein was calculated by[(IC50 of SLT-1A control protein/IC50 of experimental protein)×100].

The inhibitory effect of both versions of αCD20scFv::SLT-1A on cell-freeprotein synthesis was strong. Multiple experiments determined that theIC₅₀ of both versions of αCD20scFv::SLT-1A was around 50 picomolar (pM).In one experiment, the IC₅₀ of αCD20scFv::SLT-1A version 1 on proteinsynthesis was about 38 pM or within 19% of the SLT-1A-only positivecontrol (Table 2). Similarly, the IC₅₀ of αCD20scFv::SLT-1A version 2 onprotein synthesis in this cell-free assay was about 58 pM or within 18%of the SLT-1A-only positive control (Table 2).

TABLE 2 Ribosome Inactivation: Representative half-maximal inhibitoryconcentrations (IC₅₀) for exemplary CD20-binding proteins CD20-BindingIC₅₀ IC₅₀ of SLT-1A-only Percent of IC₅₀ of Protein (pM) positivecontrol (pM) SLT-1A control αCD20scFv::SLT- 38.3 31.2 81% 1A version 1αCD20scFv::SLT- 58.3 47.8 82% 1A version 2

Example 4—Determining Cellular Internalization by ImmunofluorescenceAssay

Immunofluorescence studies were carried out in order to analyze thebinding and internalization profiles of αCD20scFv::SLT-1A version 1 inCD20+ positive cell lines (Daudi, Raji, and Ramos) as compared toCD20-cell lines (BC-1, Jurkat (J45.01), and U266). For example, 50 nM ofthe respective CD 20-binding proteins were incubated with 0.8×10⁶ Rajicells for 1 hour at 37° C. to allow for binding and internalization ofthe CD20-binding protein. The cells were then washed with 1×PBS, fixedand permeabilized with BD cytofix/cytoperm (BD Biosciences, San Jose,Calif., U.S.), and then washed twice with 1× BD Perm/Wash™ Buffer (BDBiosciences, San Jose, Calif., U.S.). The cells were incubated withAlexa Fluor®-555 labeled mouse anti-SLT-1A antibody (BEI Resources,Manassas, Va., U.S.) in 1× BD Perm/Wash™ Buffer for 45 minutes at roomtemperature. Cells were then washed and fixed with BD cytofix (BDBiosciences, San Jose, Calif., U.S.) for 10 minutes at 4° C. The cellswere then washed with 1×PBS and resuspended in 1×PBS, and then the cellswere allowed to adhere onto poly-L-lysine coated glass slides (VWR,Radnor, Pa., U.S.). Slides were coverslipped with4′,6-diamidino-2-phenylindole (DAPI)-containing Vectashield (FisherScientific, Waltham, Mass., U.S.) and viewed by Zeiss FluorescenceMicroscope (Zeiss, Thornwood, N.Y., U.S.).

Immunofluorescence studies showed that αCD20scFv::SLT-1A version 1 andB9E9-SLT-1A bound to cell surfaces and entered into cells expressingCD20 within one hour at 37° C.

Example 5—CD20+ Cell Kill Assay: Determining the Cytotoxic Selectivityand Half-Maximal Cytotoxic Concentrations (CD₅₀) of CD20-BindingProteins

The cytotoxicity profiles of both versions of αCD20scFv::SLT-1A weredetermined by a CD20+ cell kill assay. This assay determines thecapacity of a CD20-binding protein to kill cells expressing CD20 on acellular surface as compared to cells that do not express the targetbiomolecule. Cells were plated (2×10³ per well) in 20 μL media in 384well plates. The αCD20scFv::SLT-1A protein to be tested was dilutedeither 5-fold or 10-fold in a 1×PBS, and 5 μL of the dilutions or buffercontrol were added to the cells. Control wells containing only mediawere used for baseline correction. The cell samples were incubated for 3days at 37° C. and in an atmosphere of 5% carbon dioxide (CO₂) with theαCD20scFv::SLT-1A to be tested or only PBS buffer. The total cellsurvival or percent viability was determined using a luminescent readoutusing the CellTiter-Glo® Luminescent Cell Viability Assay (G7573 PromegaMadison, Wis., U.S.) according to the manufacturer's instructions. The“percent viability” of experimental wells was calculated using thefollowing equation: (Test RLU−Average Media RLU)/(Average CellsRLU−Average Media RLU)*100. Log polypeptide concentration versus PercentViability was plotted using Prism software (GraphPad Prism, San Diego,Calif., U.S.) and log (inhibitor) vs. normalized response (variableslope) analysis was used to determine the half-maximal cytotoxicconcentration (CD₅₀) value for the exemplary CD20-binding proteins. Inaddition, cell samples from lymphoma patients were analyzed using thiscell kill assay to determine the cytotoxicity profile ofαCD20scFv::SLT-1A version 1.

Over multiple experiments, both versions of αCD20scFv::SLT-1Ademonstrated CD20-specific cell kill with 10 to 1000-fold specificitycompared to cell kill of CD20 negative cell lines (Table 3). TheCD20-specific cell kill profile of both versions of αCD20scFv::SLT-1Aalso contrasted to the ability of the component SLT-1A (251) to killcells which lacked CD20-specificity (Table 3). The CD₅₀ values of bothversions of αCD20scFv::SLT-1A protein was measured to be about 3-70 nMfor CD20+ cells, depending on the cell line, as compared to over600-2,000 for CD20-cell lines (Table 3). The CD₅₀ of theαCD20scFv::SLT-1A version 1 CD20-binding protein was over 100 to 400fold greater (less cytotoxic) for cells which did not express CD20 on acellular surface as compared to cells expressing CD20 on a cellularsurface. The CD₅₀ of αCD20scFv::SLT-1A version toward human lymphomacells from patient samples was about 7-40 nM (Table 3).

TABLE 3 Selective Cytotoxicity: Representative half-maximal cytotoxicconcentrations (CD₅₀) for exemplary CD20-binding proteins CD₅₀ (nM)SLT-1A only CD20 αCD20scFv::SLT- αCD20scFv::SLT- negative Cell Linestatus 1A version 1 1A version 2 control Daudi positive 5.6 67.0 650Raji positive 2.8 4.5 1,100 ST486 positive 3.7 7.0 940 Ramos positive27.0 33.0 470 BC-1 negative 2,000 2,100.0 160 Jurkat negative 1,400600.0 120 U226 negative 2,500 not determined 960 Patient Samplesfollicular positive 7.1 39.0 690,000 lymphoma, rituximab refractoryBurkitt's positive 9.0 12.0 960 lymphoma transformed by Epstein- BarrVirus

Example 6—Comparative CD20+ Cell Kill: Determining the RelativeCytotoxicities of CD20-Binding Proteins to CD20+ Cells

Three potentially cytotoxic CD20-binding proteins were tested using theCD20+ cell kill assay in Raji cells (CD20+) as described above inExample 5. A set of representative results is reported in Table 4. Overmultiple experiments, αCD20scFv::SLT-1A version 1 exhibited a 50 to100-fold greater cell kill function as compared to the CD20-bindingprotein B9E9 (SEQ ID NO:12) (Table 4).

TABLE 4 Representative Half-Maximal Cytotoxic Concentrations (CD₅₀) forExemplary CD20-Binding Proteins to CD20+ Raji Cells CD20-Binding ProteinCD₅₀ (nM) SLT-1A only negative control 429 αCD20scFv::SLT-1A version 1 2αCD20scFv-B9E9::SLT-1A 103

Example 7—Determining the Targeted Cytotoxicity for CD20-BindingProteins Using In Vivo Xenograft Studies

Two xenograft model systems based on an immuno-compromised mouse strainswere used to study the ability of exemplary CD20-binding proteins tokill CD20+ tumor cells in vivo and in a tumor environment over time andfor various dosages. These xenograft model systems rely onwell-characterized mouse strains that lack graft versus host responses,among other immune system deficiencies. First, an intravenous tumormodel was studied using SCID (severe combined immune deficiency) mice tocreate disseminated tumors throughout the mice in order to test the invivo effects of exemplary CD20-binding proteins on human tumor cells.Second, a subcutaneous tumor model was studied using BALBc/nude mice tocreate subcutaneous tumors on the mice, again in order to test the invivo effects of exemplary CD20-binding proteins on human tumor cells.

For the first xenograft system, thirty-two C.B.-17 SCID mice (in fourgroups of eight animals) were challenged with 1×10⁷ Raji-luc humanlymphoma derived cells (Molecular Imaging, Ann Arbor, Mich., U.S.) in200 μL PBS. On days 5-9 and 12-16 following tumor challenge, thefollowing groups received the following through intravenousadministration: Group 1: PBS; Group 2: αCD20scFv::SLT-1A version 2 at adose of 2 mg/kg; Group 3: αCD20scFv::SLT-1A version 1 at a dose of 2mg/kg; and Group 4: αCD20scFv::SLT-1A version 1 at a dose of 4 mg/kg(days 5-9 only). Bioluminescence, in 1×10⁶ photons/second units (p/s),was measured on days 5, 10, 15, and 20 using a Caliper IVIS 50 opticalimaging system (Perkin Elmer, Waltham, Mass., U.S.). FIG. 2 shows howboth versions of αCD20scFv::SLT-1A, and αCD20scFv::SLT-1A version 1 atboth dosage levels, resulted in statistically significant less totalbioluminescence compared to the PBS control. The decrease in totalbioluminescence was reflective of statistically significant reductionsin disseminated tumor burdens after treatment with a CD20-bindingprotein of the invention. FIG. 3 indicates a statistically significantincrease in survival with administration of either version ofαCD20scFv::SLT-1A. The mean survival age was increased by five days withall treatments compared to the PBS negative control.

For the second xenograft model, twenty-eight BALBc/nude (in four groupsof six or seven animals) were challenged subcutaneously with 2.5×10⁶Raji human lymphoma cells (Washington Biotechnology, Simpsonville, Md.,U.S.). Tumor volume was determined using standard methods known in theart utilizing calipers. Day 0 was set at the point when the mean tumorvolume for each mouse reached approximately 160 mm³ (one mouse from eachgroup had a tumor greater than 260 mm³ so it was excluded). On days 0-4and 7-11 the groups received intravenous administration of the followingby group: Group 1: PBS; Group 2: αCD20scFv::SLT-1A version 2 at a doseof 2 mg/kg; Group 3: αCD20scFv::SLT-1A version 1 at a dose of 2 mg/kg;Group 4: αCD20scFv::SLT-1A version 1 at a dose of 4 mg/kg. Tumor volumewas measured and graphed as a function of day of study. FIG. 4demonstrates how treatment with αCD20scFv::SLT-1A version 1 (at bothdosage levels) resulted in significantly reduced tumor volume comparedto the PBS control through to Day 24. This is also reflected in thetumor free mouse number through Day 54, reported in Table 5.

TABLE 5 Elimination of Tumors by Exemplary CD20-Binding Proteins in aSubcutaneous-Tumor Mouse Model Tumor Free Mice/ Group Total Mice PBS 0/7αCD20scFv::SLT-1A version 2, 2 mg/kg 6/7 αCD20scFv::SLT-1A version 1, 2mg/kg 5/6 αCD20scFv::SLT-1A version 1, 4 mg/kg 6/7

Example 8—Determining In Vivo Effects of a CD20-Binding Protein inNon-Human Primates

The exemplary CD20-binding protein αCD20scFv::SLT-1A version 1 wasadministered to non-human primates in order to test for in vivo effects.In vivo depletion of peripheral blood B lymphocytes in cynomolgusprimates was observed after parenteral administration of different dosesof αCD20scFv::SLT-1A version 1.

In one experiment, ten cynomolgus primates were intravenously injectedwith PBS or αCD20scFv::SLT-1A version 1 at different doses (50, 150, and450 micrograms drug/kilogram body weight (mcg/kg)) on alternative daysfor 2 weeks. Then, peripheral blood samples collected prior to dosing ondays 3 and 8 were analyzed for the percentage of B-lymphocytes whichexpressed CD20 (FIGS. 5 and 6). In cynomolgus monkeys, two distinctB-cell subsets have been described by flow-cytometry: (1) CD21 negative,CD40 positive cells which express high levels of CD20, and (2) CD21positive and CD40 positive cells which express lower levels of CD20(Vugmeyster Y et al., Cytometry 52: 101-9 (2003)). Dose-dependent B-celldepletion as compared to baseline levels from blood samples collectedprior to treatment was observed on day 3 (4, 14 and 45% decrease inanimals dosed at 50, 150 and 450 mcg/kg) and day 8 (32, 52 and 75%decrease in animals dosed at 50, 150 and 450 mcg/kg) (Table 6). Thisexperiment showed that αCD20scFv::SLT-1A version 1 was capable ofkilling CD20 positive, primate B-cells in vivo.

TABLE 6 CD20-Binding Protein Dose Dependent B- Cell Depletion inNon-Human Primates Percent Decrease in Percent Decrease in CD40+, CD20+Cells (%) CD21+, CD40+, CD20+ Cells (%) 50 150 450 50 150 450 Day mcg/kgmcg/kg mcg/kg mcg/kg mcg/kg mcg/kg 3 38 57 69 4 14 45 8 65 81 86 32 5275

Example 9—A CD20-Binding Protein Derived from the A Subunit ofShiga-Like Toxin-1 and the Antibody Ofatumumab

In this example, the Shiga toxin effector region is derived from the Asubunit of Shiga-like Toxin 1 (SLT-1A). An immunoglobulin-type bindingregion αCD20 is derived from the monoclonal antibody ofatumumab (GuptaI, Jewell R, Ann N Y Acad Sci 1263: 43-56 (2012)) which comprises animmunoglobulin-type binding region capable of binding human CD20.

Construction, Production, and Purification of the CD20-Binding ProteinSLT-1A::αCD20

The immunoglobulin-type binding region αCD20 and Shiga toxin effectorregion are linked together to form a protein. For example, a fusionprotein is produced by expressing a polynucleotide encoding theCD20-binding protein SLT-1A::αCD20. Expression of the SLT-1A::αCD20CD20-binding protein is accomplished using either bacterial and/orcell-free, protein translation systems as described in the previousexamples.

Determining the In Vitro Characteristics of the CD20-Binding ProteinSLT-1A::αCD20

The binding characteristics of the CD20-binding protein of this examplefor CD20+ cells and CD20− cells is determined by a fluorescence-based,flow-cytometry assay as described above in the previous examples. TheB_(max) for SLT-1A::αCD20 binding to CD20+ cells is measured to beapproximately 50,000-200,000 MFI with a K_(D) within the range of0.01-100 nM, whereas there is no significant binding to CD20-cells inthis assay.

The ribosome inactivation capabilities of the SLT-1A::αCD20 CD20-bindingprotein is determined in a cell-free, in vitro protein translation asdescribed above in the previous examples. The inhibitory effect of theCD20-binding protein of this example on cell-free protein synthesis issignificant. The IC₅₀ of SLT-1A::αCD20 on protein synthesis in thiscell-free assay is approximately 0.1-100 pM.

Determining the Cytotoxicity of the CD20-Binding Protein SLT-1A::αCD20Using a Cell-Kill Assay

The cytotoxicity characteristics of SLT-1A::αCD20 are determined by thegeneral cell-kill assay as described above in the previous examplesusing CD20+ cells. In addition, the selective cytotoxicitycharacteristics of SLT-1A::αCD20 are determined by the same generalcell-kill assay using CD20-cells as a comparison to the CD20+ cells. TheCD₅₀ of the CD20-binding protein of this example is approximately0.01-100 nM for CD20+ cells depending on the cell line. The CD₅₀ of theCD20-binding protein is approximately 10-10,000 fold greater (lesscytotoxic) for cells not expressing CD20 on a cellular surface ascompared to cells which do express CD20 on a cellular surface.

Determining the In Vivo Effects of the CD20-Binding ProteinSLT-1A::αCD20 Using Animal Models

Animal models are used to determine the in vivo effects of theCD20-binding protein SLT-1A::αCD20 on neoplastic cells. Various micestrains are used to test the effect of the CD20-binding protein afterintravenous administration on xenograft tumors in mice resulting fromthe injection into those mice of human neoplastic cells which expressCD20 on their cell surfaces. Non-human primates may be used to test theeffect of SLT-1A::αCD20 on peripheral blood B-cells as described abovein Example 8.

Example 10—CD20-Binding Proteins Based on Various CD20 Binding Domains

In this example, the Shiga toxin effector region is derived from the Asubunit of Shiga-like Toxin 1 (SLT-1A), Shiga toxin (StxA), and/orShiga-like Toxin 2 (SLT-2A). An immunoglobulin-type binding region isderived from the immunoglobulin domain from the molecule chosen fromTable 7 and which binds an extracellular part of CD20. The exemplarycytotoxic proteins of this example are created and tested as describedin the previous examples using CD20+ cells expressing CD20 to a cellularsurface.

TABLE 7 Exemplary CD20 Binding Domains Source of CD20 Binding Domainibritumomab obinutuzumab ocaratuzumab ocrelizumab obinutuzumabofatumumab rituximab tositumomab ublituximab CD20 binding scFv(s) inGeng S et al., Cell Mol Immunol 3: 439-43 (2006) CD20 binding scFv(s) inOtafesn T et al, Protein Eng Des Sel 23: 243-9 (2010)

While certain embodiments of the invention have been described by way ofillustration, it will be apparent that the invention may be put intopractice with many modifications, variations and adaptations, and withthe use of numerous equivalents or alternative solutions that are withinthe scope of persons skilled in the art, without departing from thespirit of the invention or exceeding the scope of the claims.

All publications, patents, and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. The U.S. provisional patent application 61/777,130 isincorporated by reference in its entirety. The complete disclosures ofall electronically available biological sequence information fromGenBank (National Center for Biotechnology Information, U.S.) for aminoacid and nucleotide sequences cited herein are each incorporated hereinby reference in their entirety.

Sequence Listings Text ID Number Description Sequence SEQ ID NO: 1Shiga-like KEFTLDFSTAKTYVDSLN Toxin 1 A VIRSAIGTPLQTISSGGT SubunitSLLMIDSGSGDNLFAVDV (SLT-1A) RGIDPEEGRFNNLRLIVE RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SGDSSYTTLQRVAGISRT GMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFV TVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILG SVALILNCHHHASRVARMASDEFPSMCPADGRVRGI THNKILWDSSTLGAILMR RTISS SEQ ID NO: 2 polynucleotideaargarttyacnytngay encoding ttywsnacngcnaaracn SLT-1 Ataygtngaywsnytnaay Subunit gtnathmgnwsngcnath (consensus)ggnacnccnytncaracn athwsnwsnggnggnacn wsnytnytnatgathgaywsnggnwsnggngayaay ytnttygcngtngaygtn mgnggnathgayccngargarggnmgnttyaayaay ytnmgnytnathgtngar mgnaayaayytntaygtnacnggnttygtnaaymgn acnaayaaygtnttytay mgnttygcngayttywsncaygtnacnttyccnggn acnacngcngtnacnytn wsnggngaywsnwsntayacnacnytncarmgngtn gcnggnathwsnmgnacn ggnatgcarathaaymgncaywsnytnacnacnwsn tayytngayytnatgwsn caywsnggnacnwsnytnacncarwsngtngcnmgn gcnatgytnmgnttygtn acngtnacngcngargcnytnmgnttymgncarath carmgnggnttymgnacn acnytngaygayytnwsnggnmgnwsntaygtnatg acngcngargaygtngay ytnacnytnaaytggggnmgnytnwsnwsngtnytn ccngaytaycayggncar gaywsngtnmgngtnggnmgnathwsnttyggnwsn athaaygcnathytnggn wsngtngcnytnathytnaaytgycaycaycaygcn wsnmgngtngcnmgnatg gcnwsngaygarttyccnwsnatgtgyccngcngay ggnmgngtnmgnggnath acncayaayaarathytntgggaywsnwsnacnytn ggngcnathytnatgmgn mgnacnathwsnwsntrr SEQ ID NO: 3linker extension GILGFVFTL for anti-CD20- scFv::SLT-1A version 2polypeptide SEQ ID NO: 4 anti-CD20-scFv:: MQVQLQQPGAELVKPGASSLT-1A version 1 VKMSCKTSGYTFTSYNVH polypeptide WVKQTPGQGLEWIGAIYPGNGDTSFNQKFKGKATLT ADKSSSTVYMQLSSLTSE DSAVYYCARSNYYGSSYVWFFDVWGAGTTVTVSSGS TSGSGKPGSGEGSQIVLS QSPTILSASPGEKVTMTCRASSSVSYMDWYQQKPGS SPKPWIYATSNLASGVPA RFSGSGSGTSYSLTISRVEAEDAATYYCQQWISNPP TFGAGTKLELKEFPKPST PPGSSGGAPKEFTLDFSTAKTYVDSLNVIRSAIGTP LQTISSGGTSLLMIDSGS GDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGF VNRTNNVFYRFADFSHVT FPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSL TTSYLDLMSHSGTSLTQS VARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRS YVMTAEDVDLTLNWGRLS SVLPDYHGQDSVRVGRISFGSINAILGSVALILNCH HHASRVAR SEQ ID NO: 5 polynucleotideatgcargtncarytncar encoding anti- carccnggngcngarytn CD20-gtnaarccnggngcnwsn scfv::SLT-1A gtnaaratgwsntgyaar version 1acnwsnggntayacntty acnwsntayaaygtncay tgggtnaarcaracnccnggncarggnytngartgg athggngcnathhtaycc nggnaayggngayacnwsnttyaaycaraarttyaa rggnaargcnacnytnac ngcngayaarwsnwsnwsnacngtntayatgcaryt nwsnwsnytnacnwsnga rgaywsngcngtntaytaytgygcnmgnwsnaayta ytayggnwsnwsntaygt ntggttyttygaygtntggggngcnggnacnacngt nacngtnwsnwsnggnws nacnwsnggnwsnggnaarccnggnwsnggngargg nwsncarathgtnytnws ncarwsnccnacnathytnwsngcnwsnccnggnga raargtnacnatgacntg ymgngcnwsnwsnwsngtnwsntayatggaytggta ycarcaraarccnggnws nwsnccnaarccntggathtaygcnacnwsnaayyt ngcnwsnggngtnccngc nmgnttywsnggnwsnggnwsnggnacnwsntayws nytnacnathwsnmgngt ngargcngargaygcngcnacntaytaytgycarca rtggathwsnaayccncc nacnttyggngcnggnacnaarytngarytnaarga rttyccnaarccnwsnac nccnccnggnwsnwsnggnggngcnccnaargartt yacnytngayttywsnac ngcnaaracntaygtngaywsnytnaaygtnathmg nwsngcnathggnacncc nytncaracnathwsnwsnggnggnacnwsnytnyt natgathgaywsnggnws nggngayaayytnttygcngtngaygtnmgnggnat hgayccngargarggnmg nttyaayaayytnmgnytnathgtngarmgnaayaa yytntaygtnacnggntt ygtnaaymgnacnaayaaygtnttytaymgnttygc ngayttywsncaygtnac nttyccnggnacnacngcngtnacnytnwsnggnga ywsnwsntayacnacnyt ncarmgngtngcnggnathwsnmgnacnggnatgca rathaaymgncaywsnyt nacnacnwsntayytngayytnatgwsncaywsngg nacnwsnytnacncarws ngtngcnmgngcnatgytnmgnttygtnacngtnac ngcngargcnytnmgntt ymgncarathcarmgnggnttymgnacnacnytnga ygayytnwsnggnmgnws ntaygtnatgacngcngargaygtngayytnacnyt naaytggggnmgnytnws nwsngtnytnccngaytaycayggncargaywsngt nmgngtnggnmgnathws nttyggnwsnathaaygcnathytnggnwsngtngc nytnathytnaaytgyca ycaycaygcnwsnmgngt ngcnmgnSEQ ID NO: 6 heavy chain CDR1 GYTFTSYNVH SEQ ID NO: 7 heavy chain CDR2AIYPGNGDTSFNQKFKG SEQ ID NO: 8 heavy chain CDR3 SNYYGSSYVWFFDVSEQ ID NO: 9 light chain CDR1 RASSSVSYMD SEQ ID NO: 10 light chain CDR2ATSNLAS SEQ ID NO: 11 light chain CDR3 QQWISNPPT SEQ ID NO: 12anti-CD2O-scFv- MQVQLVQSGAELVKPGAS B9E9::SLT-1A VKMSCKASGYTFTSYNMHpolypeptide WVKQTPGQGLEWIGAIYP GNGDTSYNQKFKGKATLT ADKSSSTAYMQLSSLTSEDSAVYYCARAQLRPNYWY FDVWGAGTTVTVSSGGGG SGGGGSGGGGSGGGGSGGGGSDIVLSQSPAILSASP GEKVTMTCRASSSVSYMH WYQQKPGSSPKPWIYATSNLASGVPARFGSGSGTSY STISRVEAEDAATYYCQQ WISNPPTFGAGTKLELKGGGGSGGKEFTLDFSTAKT YVDSLNVIRSAIGTPLQT ISSGGTSLLMIDSGSGDNLFAVDVRGIDPEEGRFNN LRLIVERNNLYVTGFVNR TNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRV AGISRTGMQINRHSLTTS YLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQI QRGFRTTLDDLSGRSYVM TAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGS INAILGSVALILNCHHHA SRVA SEQ ID NO: 13 polynucleotideatgcargtncarytngtn encoding anti- carwsnggngcngarytn CD20-scFv-gtnaarccnggngcnwsn B9E9::SLT-1A gtnaaratgwsntgyaar (consensus)gcnwsnggntayacntty acnwsntayaayatgcay tgggtnaarcaracnccnggncarggnytngartgg athggngcnathtayccn ggnaayggngayacnwsntayaaycaraarttyaar ggnaargcnacnytnacn gcngayaarwsnwsnwsnacngcntayatgcarytn wsnwsnytnacnwsngar gaywsngcngtntaytaytgygcnmgngcncarytn mgnccnaaytaytggtay ttygaygtntggggngcnggnacnacngtnacngtn wsnwsnggnggnggnggn wsnggnggnggnggnwsnggnggnggnggnwsnggn ggnggnggnwsnggnggn ggnggnwsngayathgtnytnwsncarwsnccngcn athytnwsngcnwsnccn ggngaraargtnacnatgacntgymgngcnwsnwsn wsngtnwsntayatgcay tggtaycarcaraarccnggnwsnwsnccnaarccn tggathtaygcnacnwsn aayytngcnwsnggngtnccngcnmgnttywsnggn wsnggnwsnggnacnwsn taywsnytnacnathwsnmgngtngargcngargay gcngcnacntaytaytgy carcartggathwsnaayccnccnacnttyggngcn ggnacnaarytngarytn aarggnggnggnggnwsnggnggnaargarttyacn ytngayttywsnacngcn aaracntaygtngaywsnytnaaygtnathmgnwsn gcnathggnacnccnytn caracnathwsnwsnggnggnacnwsnytnytnatg athgaywsnggnwsnggn gayaayytnttygcngtngaygtnmgnggnathgay ccngargarggnmgntty aayaayytnmgnytnathgtngarmgnaayaayytn taygtnacnggnttygtn aaymgnacnaayaaygtnttytaymgnttygcngay ttywsncaygtnacntty ccnggnacnacngcngtnacnytnwsnggngaywsn wsntayacnacnytncar mgngtngcnggnathwsnmgnacnggnatgcarath aaymgncaywsnytnacn acnwsntayytngayytnatgwsncaywsnggnacn wsnytcanncarwsngtn gcnmgngcnatgytnmgnttygtnacngtnacngcn gargcnytnmgnttymgn carathcarmgnggnttymgnacnacnytngaygay ytnwsnggnmgnwsntay gtnatgacngcngargaygtngayytnacnytnaay tggggnmgnytnwsnwsn gtnytnccngaytaycayggncargaywsngtnmgn gtnggnmgnathwsntty ggnwsnathaaygcnathytnggnwsngtngcnytn athytnaaytgycaycay caygcnwsnmgngtngcn mgnSEQ ID NO: 14 anti-CD20-scFv- MQVQLQQPGAELVKPGAS C2B8::SLT-1AVKMSCKASGYTFTSYNMH polypeptide WVKQTPGRGLEWIGAIYP GNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSE DSAVYYCARSTYYGGDWY FNVWGAGTTVTVSAGSTSGSGKPGSGEGSTKGQIVL SQSPAILSASPGEKVTMT CRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVP VRFSGSGSGTSYSLTISR VEAEDAATYYCQQWTSNPPTFGGGTKLEIKEFPKPS TPPGSSGGAPKEFTLDFS TAKTYVDSLNVIRSAIGTPLQTISSGGTSLLMIDSG SGDNLFAVDVRGIDPEEG RFNNLRLIVERNNLYVTGFVNRTNNVFYRFADFSHV TFPGTTAVTLSGDSSYTT LQRVAGISRTGMQINRHSLTTSYLDLMSHSGTSLTQ SVARAMLRFVTVTAEALR FRQIQRGFRTTLDDLSGRSYVMTAEDVDLTLNWGRL SSVLPDYHGQDSVRVGRI SFGSINAILGSVALILNC HHHASRVARSEQ ID NO: 15 anti-CD20-scFv- atgcargtncarytncar C2B8::SLT-1Acarccnggngcngarytn polynucleotide gtnaarccnggngcnwsn (consensus)gtnaaratgwsntgyaar gcnwsnggntayacntty acnwsntayaayatgcaytgggtnaarcaracnccn ggnmgnggnytngartgg athggngcnathtayccnggnaayggngayacnwsn tayaaycaraarttyaar ggnaargcnacnytnacngcngayaarwsnwsnwsn acngcntayatgcarytn wsnwsnytnacnwsngargaywsngcngtntaytay tgygcnmgnwsnacntay tayggnggngaytggtayttyaaygtntggggngcn ggnacnacngtnacngtn wsngcnggnwsnacnwsnggnwsnggnaarccnggn wsnggngarggnwsnacn aarggncarathgtnytnwsncarwsnccngcnath ytnwsngcnwsnccnggn garaargtnacnatgacntgymgngcnwsnwsnwsn gtnwsntayathcaytgg ttycarcaraarccnggnwsnwsnccnaarccntgg athtaygcnacnwsnaay ytngcnwsnggngtnccngtnmgnttywsnggnwsn ggnwsnggnacnwantay wsnytnacnathwsnmgngtngargcngargaygcn gcnacntaytaytgycar cartggacnwsnaayccnccnacnttyggnggnggn acnaarytngarathaar garttyccnaarccnwsnacnccnccnggnwsnwsn ggnggngcnccnaargar ttyacnytngayttywsnacngcnaaracntaygtn gaywsnytnaaygtnath mgnwsngcnathggnacnccnytncaracnathwsn wsnggnggnacnwsnytn ytnatgathgaywsnggnwsnggngayaayytntty gcngtngaygtnmgnggn athgayccngargarggnmgnttyaayaayytnmgn ytnathgtngarmgnaay aayytntaygtnacnggnttygtnaaymgnacnaay aaygtnttytaymgntty gcngayttywsncaygtnacnttyccnggnacnacn gcngtnacnytnwsnggn gaywsnwsntayacnacnytncarmgngtngcnggn athwsnmgnacnggnatn carathaaymgncaywsnytnacnacwsntayytng ayytnatgwsncaywsng gnacnwsnytnacncarwsngtngcnmgngcnatgy tnmgnttygtnacngtna cngcngargcnytnmgnttymgncarathcarmgng gnttymgnacnacnytng aygayytnwsnggnmgnwsntaygtnatgacngcng argaygtngayytnacny tnaaytggggnmgnytnwsnwsngtnytnccngayt aycayggncargaywsng tnmgngtnggnmgnathwsnttyggnwsnathaayg cnathytnggnwsngtng cnytnathytnaaytgycaycaycaygcnwsnmgng tngcnmgn SEQ ID NO: 16 anti-CD20-scFv::MQVQLQQPGAELVKPGAS SLT-1A version 2 VKMSCKTSGYTFTSYNVH polypeptideWVKQTPQGQLEWIGAIYP GNGDTSFNQKFKGKATLT ADKSSSTVYMQLSSLTSEDSAVYYCARSNYYGSSYV WFFDVWGAGTTVTVSSGS TSGSGKPGSGEGSQIVLSQSPTILSASPGEKVTMTC RASSSVSYMDWYQQKPGS SPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRV EAEDAATYYCQQWISNPP TFGAGTKLELKEFPKPSTPPGSSGGAPGILGFVFTL KEFTLDFSTAKTYVDSLN VIRSAIGTPLQTISSGGTSLLMIDSGSGDNLFAVDV RGIDPEEGRFNNLRLIVE RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SGDSSYTTLQRVAGISRT GMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFV TVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILG SVALILNCHHHASRVAR SEQ ID NO: 17Polynucleotide atgcargtncarytncar encoding anti- carccnggngcngarytnCD20-scFv:: gtnaarccnggngcnwsn SLT-1A gtnaaratgwsntgyaar version 2acnwsnggntayacntty (consensus) acnwsntayaaygtncay tgggtnaarcaracnccnggncarggnytngartgg athggngcnathtayccn ggnaayggngayacnwsnttyaaycaraarttyaar ggnaargcnacnytnacn gcngayaarwsnwsnwsnacngtntayatgcarytn wsnwsnytnacnwsngar gaywsngcngtntaytaytgygcnmgnwsnaaytay tayggnwsnwsntaygtn tggttyttygaygtntggggngcnggnacnacngtn acngtnwsnwsnggnwsn acnwsnggnwsnggnaarccnggnwsnggngarggn wsncarathgtnytnwsn carwsnccnacnathytnwsngcnwsnccnggngar aargtnacnatgacntgy mgngcnwsnwsnwsngtnwsntayatggaytggtay carcaraarccnggnwsn wsnccnaarccntggathtaygcnacnwsnaayytn gcnwsnggngtnccngcn mgnttywsnggnwsnggnwsnggnacnwsntaywsn ytnacnathwsnmgngtn gargcngargaygcngcnacntaytaytgycarcar tggathwsnaayccnccn acnttyggngcnggnacnaarytngarytnaargar ttyccnaarccnwsnacn ccnccnggnwsnwsnggnggngcnccnggnathytn ggnttygtnttyacnytn aargarttyacnytngayttywsnacngcnaaracn taygtngaywsnytnaay gtnathmgnwsngcnathggnacnccnytncaracn athwsnwsnggnggnacn wsnytnytnatgathgaywsnggnwsnggngayaay ytnttygcngtngaygtn mgnggnathgayccngargarggnmgnttyaayaay ytnmgnytnathgtngar mgnaayaayytntaygtnacnggnttygtnaaymgn acnaayaaygtnttytay mgnttygcngayttywsncaygtnacnttyccnggn acnacngcngtnacnytn wsnggngaywsnwsntayacnacnytncarmgngtn gcnggnathwsnmgnacn ggnatgcarathaaymgncaywsnytnacnacnwsn tayytngayytnatgwsn caywsnggnacnwsnytnacncarwsngtngcnmgn gcnatgytnmgnttygtn acngtnacngcngargcnytnmgnttymgncarath carmgnggnttymgnacn acnytngaygayytnwsnggnmgnwsntaygtnatg acngcngargaytngayy tnacnytnaaytggggnmgnytnwsnwsngtnytnc cngaytaycayggncarg aywsngtnmgngtnggnmgnathwsnttyggnwsna thaaygcnathytnggnw sngtngcnytnathytnaaytgycaycaycaygcnw snmgngtngcnmgn SEQ ID NO: 18 linker (218)GSTSGSGKPGSGEGS SEQ ID NO: 19 Strep-tag ® WSHPQFEK SEQ ID NO: 20murine hinge EFPKPSTPPGSSGGAP (murine IgG3) SEQ ID NO: 21heavy chain CDR1 GYTFTSYNMH SEQ ID NO: 22 heavy chain CDR2AIYPGNGDTSYNQKFKG SEQ ID NO: 23 heavy chain CDR3 AQLRPNYWYFDVSEQ ID NO: 24 light chain CDR1 RASSSVSYMH SEQ ID NO: 25 Shiga toxin  KEFTLDFSTAKTYVDSLN Subunit A VIRSAIGTPLQTISSGGT (StxA)SLLMIDSGTGDNLFAVDV RGIDPEEGRFNNLRLIVE RNNLYVTGFVNRTNNVFYRFADFSHVTFPGTTAVTL SGDSSYTTLQRVAGISRT GMQINRHSLTTSYLDLMSHSGTSLTQSVARAMLRFV TVTAEALRFRQIQRGFRT TLDDLSGRSYVMTAEDVDLTLNWGRLSSVLPDYHGQ DSVRVGRISFGSINAILG SVALILNCHHHASRVARMASDEFPSMCPADGRVRGI THNKILWDSSTLGAILMR RTISS SEQ ID NO: 26 Shiga-likeDEFTVDFSSQKSYVDSLN toxin 2 SIRSAISTPLGNISQGGV Subunit ASVSVINHVLGGNYISLNV (SLT-2A) RGLDPYSERFNHLRLIME RNNLYVAGFINTETNIFYRFSDFSHISVPDVITVSM TTDSSYSSLQRIADLERT GMQIGRHSLVGSYLDLMEFRGRSMTRASSRAMLRFV TVIAEALRFRQIQRGFRP ALSEASPLYTMTAQDVDLTLNWGRISNVLPEYRGEE GVRIGRISFNSLSAILGS VAVILNCHSTGSYSVRSVSQKQKTECQIVGDRAAIK VNNVLWEANTIAALLNRK PQDLTEPNQ SEQ ID NO: 27heavy chain CDR3 STYYGGDWYFNV SEQ ID NO: 28 light chain CDR1 RASSSVSYIHSEQ ID NO: 29 light chain CDR3 QWTSNPPT

The invention is claimed as follows:
 1. A CD20-binding proteincomprising the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO:12.
 2. The CD20-binding protein of claim 1, comprising the amino acidsequence shown in SEQ ID NO:
 4. 3. A CD20-binding protein consistingessentially of the amino acid sequence shown in SEQ ID NO: 4 or SEQ IDNO:
 12. 4. The CD20-binding protein of claim 3, consisting essentiallyof the polypeptide represented SEQ ID NO:
 4. 5. A pharmaceuticalcomposition comprising (i) a CD20-binding protein comprising the aminoacid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 12; and (ii) at leastone pharmaceutically acceptable excipient or carrier.
 6. Thepharmaceutical composition of claim 5, wherein the excipient or carrieris a physiological acceptable: solvent, dispersion medium, coating,antimicrobial agent, isotonic agent, absorption delaying agent, aqueoussolution, or powder.
 7. The pharmaceutical composition of claim 5, whichfurther comprises a pharmaceutically acceptable solvent, vehicle,aqueous solution, buffer, powder, surfactant, antioxidant, chelatingagent, antimicrobial agent, preservative, isotonic agent, tonicityadjusting agent, dispersion medium, coating, adjuvant, wetting agent,emulsifying agent, dispersing agent, absorption delaying agent,stabilizer, or additive.
 8. The pharmaceutical composition of claim 5,which comprises acetate, acetic acid, alcohol, alpha-tocopherol,aluminum monostearate, antibacterial agent, antifungal agent, ascorbicacid, ascorbyl palmitate, benzyl alcohol, butylated hydroxyanisole,butylated hydroxytoluene, chlorobutanol, citrate, citric acid, cysteinehydrochloride, dextrose, ethanol, ethylenediaminetetraacetic acid,ethyloleate, fixed oil, gelatin, glycerine, glycerol, lactic acid,lecithin, liquid polyethylene glycol, mannitol, methyl parabens, metalchelating agent, monostearate salt, organic ester, paraben, phosphate,phosphoric acid, polyalcohol, polyethylene glycol, polyol, propyleneglycol, propylgallate, Ringer's solution, saline, small-molecularorganic species, sodium bisulfate, sodium bisulfite, sodium chloride,sodium metabisulfite, sodium sulfite, sorbitol, sugar, syntheticsolvent, tartaric acid, vegetable oil, or water.
 9. The pharmaceuticalcomposition of claim 5, which comprises citrate, propylene glycol, andsugar.
 10. The pharmaceutical composition of claim 5, which comprisescitrate, propylene glycol, sorbitol, and water.
 11. The pharmaceuticalcomposition of claim 5, is formulated for parenteral administration,oral administration, rectal administration, nasal administration,subcutaneous administration, intramuscular administration, intravenousadministration, intradermal administration, or transdermaladministration.
 12. The pharmaceutical composition of claim 5, whichcomprises a solvate, salt, ester, or amide of the CD20-binding protein.13. The pharmaceutical composition of any one of claims 5-10, 11-12,which comprises the CD20-binding protein comprising the amino acidsequence shown in SEQ ID NO:
 4. 14. A pharmaceutical compositioncomprising (i) a CD20-binding protein consisting essentially of theamino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 12; and (ii) atleast one pharmaceutically acceptable excipient or carrier.
 15. Thepharmaceutical composition of claim 14, wherein the excipient or carrieris a physiological acceptable: solvent, dispersion medium, coating,antimicrobial agent, isotonic agent, absorption delaying agent, aqueoussolution, or powder.
 16. The pharmaceutical composition of claim 14,which further comprises a pharmaceutically acceptable solvent, vehicle,aqueous solution, buffer, powder, surfactant, antioxidant, chelatingagent, antimicrobial agent, preservative, isotonic agent, tonicityadjusting agent, dispersion medium, coating, adjuvant, wetting agent,emulsifying agent, dispersing agent, absorption delaying agent,stabilizer, or additive.
 17. The pharmaceutical composition of claim 14,which comprises acetate, acetic acid, alcohol, alpha-tocopherol,aluminum monostearate, antibacterial agent, antifungal agent, ascorbicacid, ascorbyl palmitate, benzyl alcohol, butylated hydroxyanisole,butylated hydroxytoluene, chlorobutanol, citrate, citric acid, cysteinehydrochloride, dextrose, ethanol, ethylenediaminetetraacetic acid,ethyloleate, fixed oil, gelatin, glycerine, glycerol, lactic acid,lecithin, liquid polyethylene glycol, mannitol, methyl parabens, metalchelating agent, monostearate salt, organic ester, paraben, phosphate,phosphoric acid, polyalcohol, polyethylene glycol, polyol, propyleneglycol, propylgallate, Ringer's solution, saline, small-molecularorganic species, sodium bisulfate, sodium bisulfite, sodium chloride,sodium metabisulfite, sodium sulfite, sorbitol, sugar, syntheticsolvent, tartaric acid, vegetable oil, or water.
 18. The pharmaceuticalcomposition of claim 14, which comprises citrate, propylene glycol, andsugar.
 19. The pharmaceutical composition of claim 14, which comprisescitrate, propylene glycol, sorbitol, and water.
 20. The pharmaceuticalcomposition of claim 14, which is formulated for oral, rectal, nasalparenteral subcutaneous, intramuscular, intravenous, intradermal, ortransdermal administration.
 21. The pharmaceutical composition of claim14, which comprises a solvate, salt, ester, or amide of the CD20-bindingprotein.
 22. The pharmaceutical composition of any one of claims 14-19,which comprises the CD20-binding protein consisting essentially of theamino acid sequence shown in SEQ ID NO:
 4. 23. A composition comprising(i) a CD20-binding protein comprising the amino acid sequence shown inSEQ ID NO: 4 or SEQ ID NO: 12; and (ii) a solvent or salt.
 24. Thecomposition of claim 23, wherein the solvent is water, alcohol, saline,small-molecular organic species, fixed oil, polyethylene glycol,glycerine, propylene glycol, ethanol, polyol, glycerol, acetic acid,lactic acid, vegetable oil, organic ester, or ethyloleate.
 25. Thecomposition of claim 23, which comprises: a vehicle, aqueous solution,buffer, surfactant, antioxidant, chelating agent, antimicrobial agent,preservative, isotonic agent, dispersion medium, coating, adjuvant,tonicity adjusting agent, wetting agent, emulsifying agent, dispersingagent, absorption delaying agent, stabilizer, or additive.
 26. Thecomposition of claim 23, which comprises: acetate, acetic acid, alcohol,alpha-tocopherol, aluminum monostearate, antibacterial agent, antifungalagent, ascorbic acid, ascorbyl palmitate, benzyl alcohol, butylatedhydroxyanisole, butylated hydroxytoluene, chlorobutanol, citrate, citricacid, cysteine hydrochloride, dextrose, ethanol,ethylenediaminetetraacetic acid, ethyloleate, fixed oil, gelatin,glycerine, glycerol, lactic acid, lecithin, liquid polyethylene glycol,mannitol, metal chelating agent, methyl parabens, monostearate salt,organic ester, paraben, phosphate, phosphoric acid, polyalcohol,polyethylene glycol, polyol, propylene glycol, propylgallate, Ringer'ssolution, saline, small-molecular organic species, sodium bisulfate,sodium bisulfite, sodium chloride, sodium metabisulfite, sodium sulfite,sorbitol, sugar, synthetic solvent, tartaric acid, vegetable oil, orwater.
 27. The composition of claim 23, which comprises citrate,propylene glycol, and sugar.
 28. The composition of claim 23, whichcomprises citrate, propylene glycol, sugar, and water.
 29. Thecomposition of claim 23, which comprises citrate, propylene glycol, andsorbitol.
 30. The composition of claim 23, which comprises citrate,propylene glycol, sorbitol, and water.
 31. The composition of any one ofclaims 23-30, which comprises the CD20-binding protein comprising theamino acid sequence shown in SEQ ID NO:
 4. 32. A composition comprising(i) a CD20-binding protein consisting essentially of the amino acidsequence shown in SEQ ID NO: 4 or SEQ ID NO: 12; and (ii) a solvent orsalt.
 33. The composition of claim 32, wherein the solvent is water,alcohol, saline, small-molecular organic species, fixed oil,polyethylene glycol, glycerine, propylene glycol, ethanol, polyol,glycerol, acetic acid, lactic acid, vegetable oil, organic ester, orethyloleate.
 34. The composition of claim 33, which comprises: avehicle, aqueous solution, buffer, surfactant, antioxidant, chelatingagent, antimicrobial agent, preservative, isotonic agent, dispersionmedium, coating, adjuvant, tonicity adjusting agent, wetting agent,emulsifying agent, dispersing agent, absorption delaying agent,stabilizer, or additive.
 35. The composition of claim 33, whichcomprises: acetate, acetic acid, alcohol, alpha-tocopherol, aluminummonostearate, antibacterial agent, antifungal agent, ascorbic acid,ascorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, butylatedhydroxytoluene, chlorobutanol, citrate, citric acid, cysteinehydrochloride, dextrose, ethanol, ethylenediaminetetraacetic acid,ethyloleate, fixed oil, gelatin, glycerine, glycerol, lactic acid,lecithin, liquid polyethylene glycol, mannitol, metal chelating agent,methyl parabens, monostearate salt, organic ester, paraben, phosphate,phosphoric acid, polyalcohol, polyethylene glycol, polyol, propyleneglycol, propylgallate, Ringer's solution, saline, small-molecularorganic species, sodium bisulfate, sodium bisulfite, sodium chloride,sodium metabisulfite, sodium sulfite, sorbitol, sugar, syntheticsolvent, tartaric acid, vegetable oil, or water.
 36. The composition ofclaim 33, which comprises citrate, propylene glycol, and sugar.
 37. Thecomposition of claim 33, which comprises citrate, propylene glycol,sugar, and water.
 38. The composition of claim 33, which comprisescitrate, propylene glycol, and sorbitol.
 39. The composition of claim33, which comprises citrate, propylene glycol, sorbitol, and water. 40.The composition of any one of claims 32-39, which comprises theCD20-binding protein consisting essentially of the amino acid sequenceshown in SEQ ID NO: 4.